CN101420964A - Compositions comprising human embryonic stem cells and theirderivatives, methods of use, and methods of preparation - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising of preparations of human embryonic stem (hES) cells and their derivatives and methods for their transplantation into the human body, wherein transplantation results in the clinical reversal of symptoms, cure, stabilization or arrest of degeneration of a wide variety of presently incurable and terminal medical conditions, diseases and disorders. The invention further relates to novel processes of preparing novel stem cell lines which are free of animal products, feeder cells, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor and conditioned media.; This invention further relates to the isolation, culture, maintenance, expansion, differentiation, storage, and preservation of such stem cells.

Description

The compositions, its using method and the preparation method that comprise human embryo stem cell and its derivant

Inventor: lucky Tasha sieve husband (Geeta Shroff)

Background of invention

Invention field

The present invention relates to contain the pharmaceutical composition that the people embryo does (hES) cell or derivatives thereof, described stem cell does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, be used for the treatment of can't cure at present, disease, disease or the imbalance end and medical science eventually.More specifically say, the present invention relates to method by implantation method treatment clinical disease and incurable disease or the disease that can't cure at present.The invention still further relates to the new method of the novel stem line of preparation, this stem line does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium.The invention still further relates to this class stem cell separation, cultivate, keep, increase, break up, storage and preservation.

Background technology

A large amount of physianthropy imbalances, disease and disease can't be cured by existing medicine, operation or implantation method at present, and they are incurable diseases in other words.

Stem cell can be divided " son " cell that produces this stem cell characteristic of maintenance, perhaps produces to begin to be divided into the more filial generation of the cell type of specialization, perhaps produces various types of daughter cells.Therefore, stem cell plays central role in normal person's g and D, and because of its inherent character, they also are ill or the regenerate potential source of stylish cell of damaged tissue.Stem cell is present in all stages of development and many (may be great majority) adult's tissue.Different from the cell quantity that the stem cell of different tissues and different developmental phases generally produces with type.According to this sorting technique, main stem cell type is embryonic stem cell, soma cell and embryonic genital cell.

After fertilization earliest period (early to 8 cell stages), all blastocytes all are myeloid-lymphoid stem cell (are that they can develop into and grow required all cells type fully, comprise embryo outside organization such as Placenta Hominis and umbilical cord).Arrive blastocyst stage after about 2-5 days.Produce inner cell mass in the ball of this 50-100 cell, they develop into idiosome.Inner cell mass accounts for 1/4th of this stage of development all cells, is the stem cell of a class uniqueness: embryonic stem cell.Embryonic stem cell has connate ability or the potential that is divided into 200 kinds of left and right sides soma types, is called as pluripotent stem cell.The hES cell produces all types of organizations in growth course ability does not confirm as yet fully, but they can not change its cytogene type or phenotype when long-term cultivation and quantity amplification, and keeps its pluripotency state under these conditions.

Behind the blastocyst stage, stem cell proportion in embryo, fetus and adult human body progressively reduces.Many among fetus and the adult (if not great majority) tissue contains stem cell, and they have the potential of the particular cell types that is divided into limited quantity on its normal position, so that play regeneration in the tissue of its normal presence.These stem cell (body stem cell) are pluripotent stem cells, compare them with embryonic stem cell and may have more limited potential, because they produce usually, but not whole human body cell type.The main source of body stem cell is fetus and adult bone marrow and Cord blood.

Embryonic stem cell is used as the important vitro system of research cell differentiation incident, drug screening, and uses the main source of used specialization noble cells as the reproducibility treatment.

The embryonic stem cell of pluripotency has the potentiality of development of the cell type that produces any differentiation.Therefore, can replace deficient cell and regeneration of diseased organ or tissue, also pass through the disease of heredity or the acquired depletion or the imbalance generation of stimulation dormancy and dying tissue treatment particular cell types by transplanting hES cell or derivatives thereof.

HES cell or derivatives thereof is transplanted to may be as solving the mode that unsatisfied medical science needs in the human body.

Though extensively think, transplant treatment that the hES cell will thoroughly change various diseases, disease and imbalance up to now, research still is limited to the preclinical study of decimal and Primate.Suspectable be observed result on the animal model can truly represent this class cell transplantation gone into human body after event.And, because the not clinical use of this idea, so do not determine and detect pharmaceutical composition, experimental program, route of administration and the dosage of stem cell as yet.And, though used clinically derived from the human stem cell of embryo's external source or cell, tissue and the organ of other species of xenotransplantation; But these attempt most of unsuccessful, or produce various weakening side effect.

The invention provides the people that the pharmaceutical composition that will contain hES cell and/or its derivant is implanted into disease, disease or the imbalance of suffering from various that can't cure at present or termination ends.

United States Patent (USP) 5,453,457 disclose a kind of compositions that comprises non-Mus mammal pluripotent cell that primordial germ cells produce and basic fibroblast growth factor, film in conjunction with steel factor, solubility steel factor and leukaemia inhibitory factor.

United States Patent (USP) 6,800,480 ask for protection a kind of compositions of not breaking up the hES cell of breeding on the extracellular matrix that is included in.

U.S. Patent application 2003/0017587 discloses to utilize and has not contained the culture medium amplification in vitro of the raising cell undifferentiated embryonic stem cell available from aborted fetus or fresh or refrigerated cleavage stage blastocyst.In case after separating, the embryonic stem cell adding is supplemented with in somatomedin, hyclone, the neure growth factor, leukaemia inhibitory factor, fibroblast growth factor, the cell culture medium of film in conjunction with steel factor, solubility steel factor or conditioned medium, does not wish to occur these complementary elements owing to may have side effects behind the implantation patient.Simultaneously, the method that is used for the treatment of heredity or clinical disease is not put down in writing in described patent application, except each potion of every kind of cell type of needs of patients.

U.S. Patent application 2004/0071665 provides the use mammalian stem cell to treat cardiopathic method.This example provides and is differentiated to form myocardium protogonocyte group, raising on the cell embryonic stem cell of cultivating, being then injected into three sites of the mouse heart that myocardial infarction takes place.

U.S. Patent application 2004/0107453 discloses acquisition, kept and has been divided into method and their application in therapeutic treatment of human stem cell.

U.S. Patent application 2005/0124003 discloses acquisition, keep method and their application in therapeutic treatment with the differentiated fetal stem cell.

Relevant especially with the present invention specifically is that embryo stem cell transplantation is shown that clearly it is used for the potential of the first-line treatment of acute SCI (McDonald etc., (1999) Nature Med.5:1410 in mouse spinal cord damage (SCI) model; Kerr etc., (2003) J.Neurosci.23:5131; Roy etc. (2004) Nature Biotechnology, 22:297; Hori etc. (2003) Stem Cells, 21:405; Harper (2004) Proc.Natl.Acad.Sci.101:7123).Although in animal model, have effect, but suspection can produce the graft-versus-host exclusive problem, may need give immunosuppressant and formation tumor and teratoma throughout one's life and delay for the approval that reappears clinical preceding safety and effect in the experimental human trial.In the face of design clinical research and relevant especially with the present invention other problem are, up to now, do not set up medication or scheme as yet, not have to study treatment effective dose, active pharmaceutical compositions or which kind of cell type or the cell that use and make up.

Therefore, the present invention seeks to exploitation and be suspended in biocompatible solution, carrier or substrate, thereby the pharmaceutical composition that is fit to human, it contains hES cell and its derivant, does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium.

Another object of the present invention is the disease that can't cure at present of exploitation treatment or the method for incurable disease.

Another object of the present invention is the method for exploitation treatment SCI (spinal cord injury).

The present composition be easy to preparation, safety, economy, effectively, be easy to transportation, can enlarge scale, have the excellent storage life-span and do not exist such as side effect such as antibody-antigen-reactive, anomalous innervation, tumor antigenicity, formation teratoma or graft-versus-host repulsions.In addition, the present invention only needs an embryo, does not therefore need people embryo without interruption.Simultaneously, therapeutic scheme of the present invention does not need to use immunosuppressant, and do not rely on the HLA typing, its effective treatment to disease, disease or imbalance did not rely on by the ethnic group of treatment target, sex or age, can not disappear and not need the administration those skilled in the art are trained in advance.Therefore, the clinical mechanism of any suitable equipment puts into practice the pharmaceutical composition treatment target according to the present invention in the world.

With the human embryo stem cell implant into body, it is very important as antibacterial, virus, Protein virus or viroid that this class cell does not contain pollution for therapeutic purposes.Adopt standard operation experiment standard such as Good Manufacturing Practice and Good Clinical Practice the risk of this pollution can be reduced to acceptable level.

Yet, risky for patients by existing cell culture processes treatment.

Principal risk is the cell culture medium component that retains in the medicine of administration of human object, therefore represents the patient that the risk of the unexpected side effect of predicting still takes place to study by " safety " of animal level.

Therefore, need to eliminate this class risk.

The feature in the embryonic stem cell cultivation source of administration of human object is accredited as has following design: it can not break up in the time expand internal breeding, keep the caryogram that verily keeps all donor features in the training period, in whole incubation, keep and be divided into entoderm, the potential of mesoderm and ectodermic derivant, can not break up when under not having the situation of exogenous factor, cultivating, do not produce teratoma, there is not immunogenicity, can not form unusual connective tissue and ectopic tissue, can act on damaged tissue, can not divide continuously in vivo, as they can procedurally carry out these behaviors in natural life cycle.Should there be pollutant in cultural method of the present invention and the cell line.

Yan Jiu major impetus is the condition of culture that these requirements are satisfied in exploitation up to now, but up to now, does not find or verify this class condition by clinical trial as yet.Specifically, research is up to now paid close attention to the elimination mice and is raised the needs of cell as culture matrix and the anti-differentiation of culture of human embryonic stem cells.Raising cell and replenish " conditioned medium " by removal provides the part of UGF to remedy also to have introduced unacceptable risk in ideal culture medium.Raising cell conditioned medium and exist following cultured human embryo tire stem cell still to keep the inherent risk that pollutes, is unacceptable risk during being implanted into human body therefore.

The other factors of the human embryo stem cell condition of culture of design administration of human is to existing and the residual query of the exogenous supplement of essential culture medium during cell amplification in the giving pharmaceutical composition.

They comprise basic fibroblast growth factor, leukaemia inhibitory factor, film is in conjunction with steel factor, the solubility steel factor, serum, albumin or albumin supplement, amino acid supplements, vitamin replenisher, transferrins or transferrins supplement, antioxidant, insulin or insulin substitution thing, precursor of collagen or precursor of collagen substitute, trace element, " conditioned medium " residue, animal product, raise cell, somatomedin, the combination of complementarity mineral, amino acid supplements and vitamin replenisher.

The residue of described other supplement is considered the unnecessary risk of transplanting in people's embryonic liver cell processes patient safety.In addition, this class factor is added to the risk that has increased environmental pollution in the culture medium, and improved the cost that stem-cell therapy is used, therefore limited its practicality in various medical conditions, disease or imbalance.

Adopted many methods to reduce this type of risk, having comprised: United States Patent (USP) and application number 5,843,780; 5,690,926; 6,642,048; 6,800,480; 5,166,065; 6,200,806; 5,453,357; 6,090,622; 6,562,619; 6,921,632,2006/0073587 and 2002/076747.

Yet, the system that these methods all do not provide a kind of generation to contain the medicine of human embryo stem cell and its derivant, described medicine may not influence the effect of human embryo stem cell and its derivant and the potentially contaminated sex factor of safety after not containing administration of human.

Therefore, another object of the present invention is that exploitation is a kind of with the state that do not break up substantially amplification hES cell and its derivant, is easy to be applied to the simplification cell culture system of the medicine of various medical conditions with generation.

More specifically say, the purpose of this invention is to provide that a kind of generation does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with the culture technique of the stem line of steel factor, solubility steel factor and conditioned medium.

Summary of the invention

The invention provides the pharmaceutical composition and hES cell and the application of its derivant in the various diseases of treatment, disease or imbalance that contain hES cell and/or its derivant, wherein described stem cell is introduced human body by insertion approach in various route of administration, local application or the damage.

The present invention also provides treatment to suffer from the end or that can't cure at present eventually disease, the method of the object of imbalance or disease, described method comprises passes through intramuscular, intravenous, the tail side, in the vitreous body, in the striatum, in the essence, in the sheath, epidural, behind the eyeball, subcutaneous, intracardiac, in the capsule, intraarticular or intrathecal injection, the epidural catheter infusion, subarachnoid block conduit infusion, intravenous infusion, pass through aerosol apparatus, spraying, the intravaginal approach, partial eye drop and auristillae treat effective dose hES cell or derivatives thereof scheme and give the scheme of hES cell and its derivant by approach in local or the damage.

Preferred use does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, vitamin replenisher, amino acid supplements, fibroblast growth factor, film be in conjunction with the hES cell or derivatives thereof of steel factor, solubility steel factor and conditioned medium, may with what avoid any pollution risk and side effect.Can obtain hES cell and its derivant by any cell culture processes known or approval, it does not contain the pollutant of raising cell and any source, and it is safe that the people is transplanted.The hES derivant comprises the cell that further breaks up from human body.

The present invention also provides the pharmaceutical composition of disease, imbalance or the disease for the treatment of the termination property or can't cure at present, it comprises hES cell and/or its derivant that is suspended in the treatment effective dose in pharmaceutically acceptable biocompatible solution or any other carrier, and wherein said hES cell or derivatives thereof does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium.

The present invention also comprises and does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, hES cell and/or its derivant of embedding in biological compatibility material or substrate.Described biocompatible materials or substrate are optional from biological polymer, comprise polypeptide or protein, polysaccharide comprises fibronectin, various types of collagen, laminin, keratin, fibrin, Fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin.

The present composition can be contained in the instant medicament forms, and wherein said stem cell has enough vigor, and promptly their vigor is enough high, for use in one or more methods of the present invention.In one embodiment, the vigor of described stem cell is greater than about 40%, as greater than about 50%, 60%, 70% or 80%.Said composition also can comprise antimicrobial, antibacterial, hormone product or other medicament.

In order to prepare said composition, with about 750,000-160,000,000 hES cell and/or one or more derivants such as hematopoietic stem cell CFU-GM, neuronal stem cell CFU-GM, mescenchymal stem cell CFU-GM, insulin-producing stem cell CFU-GM, hepatocyte stem cell CFU-GM, cardiac stem cells CFU-GM, epithelial stem cell CFU-GM or its mixture are suspended in about 0.25-100ml carrier.In one embodiment, with about 750,000-80,000,000 hES cell suspension is in about 0.25-10ml carrier.The stem cell CFU-GM type of the specific differentiation of priority enrichment in the colony, but will keep the undifferentiated stem cell of certain proportion in the compositions.In one embodiment, the ratio of undifferentiated stem cell is no more than about 80% in whole cell colonys.In another embodiment, the ratio of undifferentiated stem cell is no more than about 40% in whole cell colonys.

Can include but not limited to other imbalance or disease according to the present invention's treatment or the termination disease of improving: cancer, hepatopathy and nephropathy, nervous system disease, dermatosis, autoimmune disease, genetic diseases, oculopathy, the flesh bone disease, fertility and reproductive disease, and cardiovascular diseases, include but not limited to: acute myeloid leukaemia, adenocarcinoma, arthritis, astrocytoma, the auditory nerve atrophy, autism, autoimmune disease, Alzheimer's disease, ankylosing spondylitis, becker's dystrophy, brain injury, burn, cerebrovascular trauma, the cerebral palsy, stupor, corneal ulcer, corneal graft rejection, neural cortex-substrate degeneration, coronary heart disease, diabetes, dull-witted, mongolism, Duchenne's dystrophy, end-stage renal disease, erb's palsy, face shoulder muscular dystrophy (Fascio Scapular MuscularDystrophy), fertility is sick, Friedreich ataxia, heart failure, hepatocarcinoma, heritability dynamoneure disease, Huntington chorea, krabbe's disease, limb-girdle muscular dystrophy, liver cirrhosis, degeneration of macula, mental retardation, multiple sclerosis, motor neuron, myocardial infarction, nephrotic syndrome, Niemann-Pick disease, the disunion skin ulcer, olivopontocerebellar atrophy, optic atrophy, parkinson disease, electric shock back encephalopathy, encephalopathy behind the rabies vaccine, decubital ulcer, progressive supranuclear plasy, psoriasis, phthisis bulbi, restrictive cardiomyopathy, retinitis pigmentosa, right bundle branch block, sarcoidosis, sinus bradycardia, spinal cord muscular dystrophy, spinocerebellar ataxia, Stevens Johnson syndrome, systemic lupus erythematosus (sle), thrombocytopenia, thalassemia, ulcerative colitis, vegetative state, cystic fibrosis, interstitial lung disease, azoospermia, primary ovarian failure, aphthous ulcer, hormonal imbalance, osteoarthritis, Horner syndrome and osteogenesis imperfecta, and passage disease and hypogammaglobulinemia.

Say that more specifically the invention provides the method for the SCI of treatment target, described method comprises:

A) give by subcutaneous injection about 750,000-80,000,000 hES cell and/or its derivant;

B) back at the fixed time repeating step (a) gives hES cell and/or its derivant by intramuscular injection then;

C) treat hES cell and/or its derivant of effective dose by intravenous injection or infusion, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM;

D) treat hES cell and/or its derivant of effective dose by epidural injection, wherein said cell comprises the neuronal stem cell CFU-GM, and described dosage is repeated according to the object situation that clinical and/or neurological detect assessment in the back at the fixed time;

E) treat hES cell and/or its derivant of effective dose by the tail side injection, wherein said cell comprises the neuronal stem cell CFU-GM;

F) treat hES cell and/or its derivant of effective dose by intrathecal injection or subarachnoid block conduit, wherein said cell comprises the neuronal stem cell CFU-GM;

G) treat hES cell and/or its derivant of effective dose by epidural injection or epidural catheter, wherein said cell comprises the neuronal stem cell CFU-GM;

H) treat hES cell and/or its derivant of effective dose at the spinal cord either side by degree of depth spinal cord injection; With

I) treat hES cell and/or its derivant of effective dose by intravenous infusion;

Step (a) and (b) at first carry out wherein, all the other steps are carried out with random order.In one embodiment, carry out step (g) after the step (f), repeat at least once, reveal the clinical indication of recovering by described SCI up to Object table.

The present invention also provides a kind of disease of suffering from, the method of the object of imbalance or disease, described method comprises by intramuscular injection, intravenous injection, epidural injection, epidural catheter, retrobulbar injection, subcutaneous injection, intracardiac injection, intracapsular injection, use hES cell and/or its derivant for the treatment of effective dose in intrathecal injection or local application or the damage, they are with not containing animal product, raise cell, conditioned medium, somatomedin, leukaemia inhibitory factor, fibroblast growth factor, cultivate in the culture medium of film in conjunction with steel factor or solubility steel factor.In one embodiment, described disease, imbalance or disease are the end or that can't cure at present eventually disease, imbalance or diseases.

The present invention also provides a kind of method for the treatment of disorder of development, degenerative disease, familial disease and traumatic nervous system disease and cerebrovascular disease, described method comprises and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM alone or in combination, by intravenous injection, subcutaneous injection, intramuscular injection, intrathecal injection, epidural catheter infusion and subarachnoid block conduit infusion.

The present invention also provides a kind of treatment dermopathic method, described method comprise give by subcutaneous or intravenous injection about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

The present invention also provides a kind of method for the treatment of decubital ulcer, described method comprise give by part or local application and intramuscular injection about 750,000-160,000,000 hES cell and/or its derivant.

The present invention also provides a kind of method for the treatment of autoimmune disease, described method comprises by intramuscular injection, intravenous injection, subcutaneous injection or intra-articular injection or intravenous infusion or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

The present invention also provides a kind of method for the treatment of genetic diseases, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intrathecal injection, epidural catheter infusion or subarachnoid block conduit infusion or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM alone or in combination.

The present invention also provides a kind of method for the treatment of gangrene, and described method comprises by intravenous injection, intramuscular injection or living and dead to organize junction local application or its combination to give about 750,000-160,000,000 hES cell and/or its derivant.

The present invention also provides the method for a kind of treatment disease relevant with aging, described method comprise with suspension or be blended in biological compatibility carrier such as gel, ointment, substrate, paste or aerosol spray in form give about 750 by intravenous injection, subcutaneous injection, intramuscular injection or local application, 000-160,000,000 hES cell and/or its derivant.

The present invention also provides a kind of method for the treatment of diabetes, described method comprise give by intravenous or intramuscular injection or its combination about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the insulin-producing CFU-GM.

The present invention also provides a kind of method for the treatment of cardiovascular disease, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intracardiac injection, angiography or the direct injection in performing the operation gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

The present invention also provides a kind of method for the treatment of hepatopathy and nephropathy, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intravenous infusion or local injection and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise the hematopoietic stem cell CFU-GM, produce albumin stem cell CFU-GM and produce bilirubin stem cell CFU-GM.

The present invention also provides a kind of method for the treatment of fertility disease or reproduction disease, described method comprise by in local intramuscular injection, the testis injection or epididymis near subcutaneous injection give about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

The present invention also provides a kind of method for the treatment of the flesh bone disease, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection or intravenous catheter infusion and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM alone or in combination.

The present invention also provides a kind of method for the treatment of oculopathy, described method comprises by local intravenous injection, subcutaneous injection, intramuscular injection, retrobulbar injection, intravitreal injection or local application and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM, hematopoietic stem cell CFU-GM and mescenchymal stem cell CFU-GM alone or in combination.In one embodiment, comprise the about 750 of neuronal stem cell CFU-GM, 000-160,000,000 hES cell and/or its derivant by retrobulbar injection.In another embodiment, comprise the about 750 of neuronal stem cell CFU-GM, 000-160,000,000 hES cell and/or its derivant by intravitreal injection.In another embodiment, to comprise about 750 of mescenchymal stem cell CFU-GM, 000-160,000,000 hES cell and/or its derivant put on adherent lens, be placed on and treat corneal abrasion (as cultivating this adherent lens,, then this eyeglass being placed on the eyeball about 24 hours) on the eyeball so that with this adherent lens of cell envelope with hES cell and/or its derivant.

The present invention also provides a kind of method for the treatment of pneumonopathy, described method comprises by intramuscular injection, intravenous injection, spraying or aerosol apparatus and gives 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise separately or with the hematopoietic stem cell CFU-GM of neuronal stem cell CFU-GM combination.In one embodiment, give 750,000-160,000,000 hematopoietic stem cell CFU-GM by the aerosol apparatus that adds the 2ml normal saline.In another embodiment, give 750,000-160,000,000 hematopoietic stem cell CFU-GM by spraying (smog).

The present invention also provides a kind of method for the treatment of the hormone disease, and described method comprises by intramuscular and intravenous route and gives 750,000-160, and 000,000 hES cell and/or its derivant, wherein said cell comprises hematopoietic stem cell and neuronal stem cell.

The present invention also provides a kind of method for the treatment of aphthous ulcer and other ulcer, described method comprises by intramuscular and intravenous route and gives 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise combination or independent hematopoietic stem cell and neuronal stem cell.

The present invention also provides a kind of method for the treatment of knee joint and coxarthrosis, and described method comprises by intramuscular, intravenous or intra-articular injection and gives 750,000-160,000,000 hES cell and/or its derivant.In one embodiment, give 750,000-160,000,000 hematopoietic stem cell CFU-GM by the intraarticular approach.

The invention provides a kind of cell culture processes that can be used for cultivating hES cell and its derivant, described hES cell can be used for transplanting with its derivant gives the people, and can not cause that any side effect is as forming teratoma, formation tumor, antigenicity problem or graft-versus-host repulsion.

The design of this cultural method is used in particular for producing and is implanted into hES cell and its derivant that can obtain active enough vigor of optimal treatment and proper characteristics behind the human body.

In a preferred embodiment, cultivate, increase, break up and store the hES cell with this cell culture processes.In addition, the present invention relates to injection-type compositions immediately, it contains hES cell and/or its derivant that stores under the condition of storage of back directly transplanting that is fit to thaw.

Substantially the existing cell culture processes of differentiation state is not opposite with the hES cell is maintained at, this culture medium does not contain supplement, as basic fibroblast growth factor, leukaemia inhibitory factor, film is in conjunction with steel factor, the solubility steel factor, serum, albumin or albumin supplement, amino acid supplements, vitamin replenisher, transferrins or transferrins supplement, antioxidant, insulin or insulin substitution thing, precursor of collagen or precursor of collagen substitute, trace element, " conditioned medium " residue, animal product, raise cell, somatomedin, the combination of complementarity mineral, amino acid supplements and vitamin replenisher.

Opposite with other cell culture processes of amplification hES cell, implement the present invention and do not cause forming embryoid, thereby avoided the excision that need undergo surgery.It should be noted that product of the present invention is people's product fully this moment, do not need to carry out xenotransplantation (detecting as animal).The conceived situation of external fertilization (IVF) result (corollary example) that is inevitable for the first time.

Unlike other cell culture processes, the cell culture technology of the present invention hES cell that can under the situation of not breaking up substantially, almost unrestrictedly increase.Major advantage of the present invention is hES cell and/or its derivant that an embryo is enough to provide the treatment effective dose, to treat a plurality of patients.Therefore, do not need to repeat to obtain the people embryo, can avoid many ethical issuess relevant with using the people embryo.

Another advantage of the inventive method is from the immunology angle, and hES cell and its derivant are generally accepted products, does not therefore need the cross-matched of patient and cell, does not need immunosuppressant.

One aspect of the present invention relates to the method for separating the hES cell, and described method comprises:

(a) with 2-7 age in days embryo collection in minimal essential medium,

(b) obtain the hES cell by mechanical means by the embryo.

One aspect of the present invention relates to that amplification does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with the method for the human embryo stem cell of steel factor, solubility steel factor and conditioned medium, said method comprising the steps of:

(a) human embryo stem cell is introduced in the cell culture medium of being made up of minimal essential medium, progestogen and β-human chorionic gonadotropin (β hCG) agonist; With

(b) under about 34 ℃-38 ℃ temperature, cultivated described stem cell about 12-48 hour in the environment of about 3.5%-6% carbon dioxide.

The present invention relates to a kind of method of the hES of cultivation cell on the other hand, this method makes cell be in part differentiation state state before, and do not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, this method may further comprise the steps:

(a) the hES cell is introduced in the cell culture medium of being made up of minimal essential medium; With

(b) under about 34 ℃-38 ℃ temperature, cultivated described stem cell about 12-48 hour in the environment of about 3.5%-6% carbon dioxide.

The present invention relates to the method that a kind of preparation is used for the human instant human embryo stem cell preparation of transplanting on the other hand:

(a) obtain not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with the human embryo stem cell of steel factor, solubility steel factor and conditioned medium

(b) centrifugal described stem cell obtain precipitation and

(c) described precipitation is suspended in biocompatible solution.

The present invention relates to the method that stores human embryo stem cell with the condition of living on the other hand, and described method comprises:

(a) get the stem cell that the inventive method prepares,

(b) add frozen dose and

(c) in-4 to-80 ℃ of freezing described cells.

The purpose of cultivating the hES cell according to the present invention's practice under the condition that does not have these class supplement is to reduce the risk of antibacterial, fungus, virus or other pollutant being introduced culture.This has reduced the risk of infection and cell characteristic variation.

Another purpose of the present invention provides and a kind ofly produces and simple, economic, the method that can enlarge scale of amplification hES cell and its derivant with the form that does not contain any contaminative residue and human safety.

Therefore, the object of the invention provides the medicine that a kind of risk that gives all common pollutant reduces and the residual contamination of recruitment factor is eliminated.

Another purpose of the present invention provides a kind of cell culture processes that reduces the risk of chromosomal aberration or genetic instability.

Another purpose of the present invention provides a kind of cell culture processes that teratoma formation risk reduces among the patient who transplants described culture that makes.

Another purpose of the present invention provides a kind of cell culture processes that tumor formation risk reduces among the patient who transplants described culture that makes.

Another purpose of the present invention provides the cell culture processes that a kind of risk that makes antigenicity problem among the patient who transplants described culture or graft-versus-host exclusive problem reduces.

The present invention provides the compositions that contains hES cell and its derivant and biocompatibility culture medium especially, and the international regulatory standard of its form and safety and quality is compatible, therefore can inject to people patient immediately and treat.

The present invention also provides a kind of hES cell that is suspended in biocompatible solution and/or goods of its derivant suspension of comprising, and these goods are included in the container so that storage, transportation or directly transplanting are given the people.

The present invention also provides the method for a kind of hES of amplification cell and its derivant.

The method that the present invention also provides a kind of hES of amplification cell and its derivant and suppresses its differentiation.

The present invention also provides the method for a kind of hES of cultivation cell and its derivant, and described method makes hES cell and its derivant suffer from disease, can effectively treat it behind the people of disease or imbalance being implanted into.

Detailed Description Of The Invention

Definition

This paper has used many terms in following description and embodiment.In order to provide to description and claims, comprise clear and definite with the consistent understanding of the given range of described term, provide to give a definition.

It may be noted that in the mode that keeps the known implication of its those of ordinary skills and use describe, in general terms term of the present invention.

" embryonic stem cell " refers to people's pluripotent cell (being the hES cell).In one embodiment, separate the hES cell by preceding blastocyst stage embryo.In another embodiment, dedifferente by making the cell (as pluripotent cell) that breaks up to small part, preparation hES cell, they are actually totipotent cell.The method for preparing the hES cell is known, referring to for example, and United States Patent (USP) 5,843,780,6,200,806,7,029,913,5,453,357,5,690,926,6,642,048,6,800,480,5,166,065,6,090,622,6,562,619,6,921,632 and 5,914,268, the U.S. openly applies for 2005/0176707 and International Application No. WO 2001085917.

" fetal stem cell " refers to by the embryo tissue, is about to the stem cell that the embryo implants the generation of people's tissue of growing behind the uterus.

" multipotency " refers to produce the stem cell of the cell of closely related cell family.

" pluripotency " refers to the stem cell as the totipotent cell offspring, can grow into any cell type except that myeloid-lymphoid stem cell.

" all-round " refers to that ovum and sperm merge the stem cell that produces.The cell that division several times produces before the fertilized egg cell also is all-round.These cells also can grow into the cell of any kind.

Be described in component such as any other derivant of embryonic stem cell, neural progenitor cell, neuronal cell, hematopoietic stem cell CFU-GM, cardiac stem cells CFU-GM or stem cell or the concentration or the consumption of other material or its mixture of implementing effectively to produce in the one or more aspects of the present invention required result with " treatment effective dose " in this description.

Use " transplanting " to describe the process that embryonic stem cell of the present invention and/or its derivant is delivered to human body in this description in the whole text, described position is need to produce beneficial effect to treat or improve disease described herein, the cell site of living in of imbalance or disease, these beneficial effects include but not limited to, repair object central nervous system's damage, treatment neurodegenerative disease or treatment apoplexy, the cardiovascular diseases, (for example causing) brain that heart attack or physical damnification or wound or genetic damage or environmental stimulus cause and/or the nerve injury of spinal cord or other organ by accident or other activity, hepatic injury, autoimmune disease, property or reproductive function obstacle, the different piece degeneration of eyes, injury of kidney etc.

Refer to wherein be suspended with hES cell and/or its derivant with " biocompatible solution " in this description, be used to transplant or the solution of any other follow-up operational version.This class biocompatible solution comprises saline, also can comprise other composition such as antiseptic, antimicrobial etc. and other pharmaceutical agents.

Refer to wherein to be suspended with solid carrier, solution and the mixture that hES cell and/or its derivant are used for topical therapeutic, transplanting or any other follow-up use with " biological compatibility carrier " in this description.This class biological compatibility carrier comprises gel, ointment, paste and aerosol spray.

In this description, " biocompatibility container " refers to wherein be equipped with hES cell and/or its derivant and can not hinder cell to be used for transplanting, and promptly do not use the container (as cell culture or storage capsule) of the compound polluted cell that can not give object.The example of biocompatibility container material includes but not limited to: glass, rustless steel and polystyrene.

" progestogen " are any progesterone sample activity that has, and promptly have at least 25% the active natural or synthetic hormone of progesterone in a kind of known Determination of biological activity.Example includes but not limited to: progesterone, dydrogesterone, medroxyprogesterone, norethindrone, levonorgestrel, norgestrel, gestodene and drospirenone.The example of other progestogen is referring to United States Patent (USP) 7,091, and 234,7,084,151,7,081,457,7,071,205,6,562,857,6,319,911,6,245,757,6,043,235 and 6,028,064.

＂ β-human chorionic gonadotropin (β hCG) agonist ＂ be defined in have the active any natural generation of at least 25% natural β hCG in a kind of known Determination of biological activity or synthetic β hCG or its fragment or derivant.The example of β hCG agonist includes but not limited to: β hCG and United States Patent (USP) 6,635,445,6,585,982,6,583,109,6,469,139 and 5,997,871 described chemical compounds.

In this manual, refer to contain the cell culture medium of aminoacid, salt, glucose and vitamin with " minimal essential medium ".Example comprises RPMI, DMEM, EMEM and GMEM.

" spraying " refer to by aerosol apparatus with medicine or substance delivery to lung.

Intraarticular refers to be administered to the intraarticular space.

Refer to be administered to the eyeball rear space behind the eyeball.

Intravenous infusion refers to that the iv liquid that will contain product or medicine feeds vein.

" epidural " injection or conduit infusion refer to be administered to the pachymeninx perimeter of meninges.

" in the sheath " injection or conduit infusion refer to be administered to the innermost layer of meninges, promptly in the cerebrospinal fluid that arachnoidea is interior with brain is communicated with.

" tail side " injection refers to by the sacrum periosteum, i.e. the coccyx top about three centimeters administrations in top, and it is connected with the epidural space.

" degree of depth spinal cord injection " pointed injection is in the erector spinae of vertebra either side.

" intramuscular " injection refers to be administered between the muscle lamella.

" intravenous " injection refers to be administered to intravenous.

" acute SCI " refers to after day of SCI three months at the most.

" subacute SCI " refers to that back three months of the day of SCI is to damaging in the future nine months at the most.

" chronic SCI " refers to that the day of SCI is back more than nine months.

" derivant " of hES cell comprises pluripotent stem cell, pluripotent stem cell, adult stem and tissue specificity stem cell, do not comprise the cell of differentiation fully.The example of tissue specificity stem cell can see table.

Cell type	The patent No.
		The stem cell of adipose-derived	6,777,231
The breast epithelium stem cell	5,814,511
		Endothelial stem cell	6,852,533
The dorsal root ganglion CFU-GM	6,835,567
		Hemopoietic progenitor cell CD34 -、CD7 +、Lin -、Lin -、 CD45RA +、	6,537,807
Hematopoietic stem cell Thy-1 +	5,061,620
		Hematopoietic stem cell Thy-1 +，CD34 +	5,750,397

Hemopoietic stem cell CD 34 +，CD38 -，HLA-DR +	5,840,580
		Hemopoietic lymph and dendritic cell CD34 +、CD45RA +、CD10 + Hemopoietic dendritic cell CD34 +、CD45RA +、CD10 +；	5,972,627
Hematopoietic stem cell c-kit -、Thy-1 -	5,876,956
		Hemopoietic stem cell CD 34 +	5,681,559
Hematopoietic stem cell HCC-1 +	5,677,136
		Hemopoietic progenitor cell CD34 +, galactose-specific agglutination element +	5,858,782
Hematopoietic stem cell (static) CD34 +	5,807,686
		The keratinocyte stem cell	6,485,971
Liver stem cells is not expressed OC2	6,129,911 6,872,389
		Lymph hemopoietic progenitor cell stem cell My10 -	5,256,560
The lymph CFU-GM	6,908,763
		The midbrain neural progenitor cell	6,913,925
Mescenchymal stem cell CD45 +	6,387,367
		Mescenchymal stem cell	5,486,359
Mescenchymal stem cell	5,827,735
		Mescenchymal stem cell	5,908,782
Mescenchymal stem cell	6,936,281
		Mescenchymal stem cell	6,908,764
Miao Leguan-deutero-pluripotency epithelial cell	6,416,999
		Myeloid progenitor c-kit hi、IL-7Rα -	6,465,247
Myeloid progenitor Thy-1 -、IL-7Rα -	6,761,883
		Neural progenitor cell	5,753,505
Neural stem cell	5,851,832
		Neuron progenitor cell	6,251,669
Neuronal stem cell	6,969,608
		Do not express the proteic neuron progenitor cell of Hu	6,852,532
The restricted neuronal precursor of pedigree	6,734,015

E-NCAM +
		Neuroepithelial stem cell	7,037,702
Central nervous system's neural stem cell	5,968,829
		Neuron progenitor cell	6,812,027
Neural progenitor cell	6,913,925
		The central nervous system neurons restrictive precursor	6,787,353
Veutro midbrain neuron CFU-GM	5,411,883
		Stem cell of neural crest	5,589,376
Contain the proteic neural crest pluripotent cell of RET	6,890,724
		The neutrophil cell precursor	5,955,357
The pancreas CFU-GM	6,436,704
		Islet progenitor cells is expressed ErbB2	6,753,153
Pluripotent cell	5,914,268
		Pluripotent cell with the HOX11 gene transformation	5,874,301
The peripheral blood CFU-GM	5,541,103
		The kidney stem cell	6,410,320
The kidney stem cell	6,458,588
		Retinal stem cells	6,117,675
The skeleton CFU-GM	6,517,872
		Stem cell FGFR +, non-ES cell	6,767,737
Produce the stem cell CD 34 of hemocyte -、MHC-I -、MHC-II -	5,744,347
		T pedigree CFU-GM CD8 int、CD4 int、c-kit hi, high bcl-2; CD8 lo、CD4 lo、c-kit hi, high bcl-2	5,821,108
T lymphocyte precursor cell CD34 +、CD7 +、Leu8 +	5,622,853

The present invention relates to be used for that clinical treatment can't be cured at present or the pharmaceutical composition that contains hES cell and/or its derivant of last disease, disease or imbalance eventually, described stem cell does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium.In another embodiment, the present invention relates to a kind ofly can't cure at present with hES cell and/or its derivatives for treatment or the whole method of last disease by implantation method.

Term " do not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film in conjunction with steel factor, solubility steel factor and conditioned medium " is not got rid of owing to cultural method of the present invention makes and is had trace progestogen and β hCG agonist in the pharmaceutical composition.Term " animal product " refers to any inhuman product.

In another embodiment, the inventive method provides a kind of with the instant form and do not contain the form of raising cell and any other pollutant and transplant simple, the safety of the pharmaceutical composition contain hES cell and/or its derivant, extensively be suitable for, can reappear and effective method.These cells can be produced by any cultural method, no matter not how unequal patient's genetic background, age, race and sex transplantation gone into human body and treated various diseases, disease and imbalance, and can not cause that antibody-antigen-reactive or graft-versus-host repel, do not form teratoma or tumor or other weakening property side effect, can not produce anomalous innervation does not need to give immunosuppressant yet.

Opposite with other known method, with do not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, therefore do not contain the hES cell of medium preparation pharmaceutical composition of the present invention of pollutant of any kind of of the safety that may disturb required clinical practice or effectiveness.

According to the present invention practice, can pass through in intramuscular, intravenous, tail side, the vitreous body, in the striatum, in the essence, in the sheath, after the epidural, eyeball, in subcutaneous, oral, intracardiac, the capsule, intraarticular or intrathecal injection or epidural catheter, subarachnoid block conduit, intravenous infusion, by aerosol apparatus, spraying, intravaginal approach and/or partial eye drop and auristillae with in hES cell and/or its derivant introducing human body.In another embodiment, give hES cell and/or its derivant by approach in local or the damage.

Each route of administration adopts the injection of designated volume, therefore adopts the cell from the specific quantity scope of hES cell and/or its derivant mother solution, and is as shown in table 1.When indicating cell type (as following infusion protocol table), the indication cell type is the main type that exists in the cell colony.

Table 1

Route of administration	Volume	Cell number
			Intramuscular	0.25ml	750,000-1,500,000
Intravenous	0.25ml	750,000-1,500,000
			Subcutaneous	0.25ml	750,000-1,500,000
The tail side	2ml	6,000,000-16,000,000
			Epidural	2ml	6,000,000-16,000,000
In the sheath	2ml	6,000,000-16,000,000
			Intraarticular	2ml	6,000,000-16,000,000
Behind the eyeball	2ml	6,000,000-16,000,000
			Epidural catheter	4-5ml	12,000,000-40,000,000
Intravenous infusion	0.75ml	2,250,000-4,500,000
			Aerosol apparatus	2ml	6,000,000-16,000,000
Intravaginal	2ml	6,000,000-16,000,000

In laboratory and final use, between clinical or other purposes, pharmaceutical composition must cold preservation.In one embodiment, pharmaceutical composition be stored in approximately+4 ℃ to-160 ℃.In another embodiment, pharmaceutical composition is stored in-15 ℃ to-72 ℃ approximately, for example about-20 ℃ to-40 ℃.

In one embodiment, the used hES cell of the present invention is to be separated, abandoned and donated by Informed Consent Form by the natural external fertilization cycle by unnecessary embryo.In another embodiment, the present invention adopts the pluripotency fetal stem cell, as the stem cell of the open application number 2005/0124003 disclosed separation of the U.S. from chorionic villus, amniotic fluid and/or Placenta Hominis.

The invention provides a kind of method with hES cell and/or its derivatives for treatment various diseases, disease and imbalance, described disease, disease and imbalance include but not limited to: cancer, apoplexy, genetic diseases, hepatopathy, nervous system disease, angiopathy, dermatosis and imbalance, autoimmune disease, oculopathy, nephropathy, heart disease, flesh bone disease, reproduction and fertility disease and arthritis.

Can comprise tumor of spinal cord according to the non-limitative example of the cancer of the present invention treatment, breast carcinoma, carcinoma of prostate, lymphoma, skin carcinoma, cancer of pancreas, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer disease, primary brain cancer, head and neck cancer, glioma, glioblastoma multiforme, hepatocarcinoma, bladder cancer, nonsmall-cell lung cancer, head and neck cancer, breast carcinoma, ovarian cancer, cervical cancer, metastasis (metastases), pulmonary carcinoma, small cell lung cancer, wilms' tumor, cervical cancer, carcinoma of testis, bladder cancer, cancer of pancreas, gastric cancer, colon cancer, carcinoma of prostate, genitourinary system carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, carcinoma of endometrium, adrenocortical carcinoma, pernicious pancreas insulinoma, the carcinoid malignant tumor, choriocarcinoma, malignant hypercalcemia, the cervix uteri hypertrophy, leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloblastic leukemia, chronic granulocytic leukemia, chronic myelocytic leukemia, acute myeloblastic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi, polycythemia vera, essential thrombocythemia, Hodgkin, non-Hodgkin lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinaemia and retinoblastoma.

In one embodiment, handle the hES cell, have embryonal cell and be programmed, in case after giving object, this cell is moved to damage location immediately without differentiation agent.At this position, this cell is corresponding and break up to differentiation signal in the body.At damage location, hES cell and its derivant can infinite multiplication, the therefore reinjected schemes of needs.HES cell and its derivant can infinite multiplication the fact eliminated formation tumor and teratomatous probability.

Put into practice with after hES cell and/or the processing of its derivant according to the present invention, the patient should have a rest and adopt the ring addiction scheme of strictness, can not take in any material such as ethanol or Nicotiana tabacum L. that possibility damaging cells function and obstruction hES cell and its derivant are moved to the regenerative process of damage location initiation.The patient also should avoid taking known to the deleterious any medicine of phenolics, because this class medicine may produce harmful effect to transplanted cells.

With hES cell and its derivatives for treatment SCI

Can't effectively cure SCI at present, comprise acute spinal cord injury, subacute spinal cord injury or chronic spinal cord lesion, they usually cause hemiplegia, quadriplegia and quadriplegia.Transplant hES cell therapy SCI and represented the unique opportunity that solves the medical need that is not met as yet, significantly improved the life that is subjected to millions of people that the SCI sequela influences in the world.Regain the central nervous system function that autonomic nervous system loses of unifying by transplanting the hES cell be the CFU-GM state basically and its derivant, can be by replacing the cell function lost, neurocyte that regeneration is lost by the hES cell CFU-GM of transplanting, eliminating physical barrier such as scar tissue, blocking-up inhibition signal transduction path and discharge neurotrophic factor and recover parasympathetic nervous, sympathetic nerve, nervus motorius, vegetative nerve and sensory nerve path.

Transplanting the hES cell according to the present invention with the benefit of recovering function of nervous system is: improve nervus motorius and sensory nerve function, improve overall quality of life, the support that provided by relatives usually is provided, reduces the dependence to other medicines compositions and other medical apparatus and instruments and adminicle, improve spirit and mental status and economic independence.By recovering parasympathetic nervous, sympathetic nerve, sensory nerve and nervus motorius function, reduce the cost of armarium and anergy auxiliary appliance and drug dependence.Improve self-sufficient ability, enhance, improve marital status and exercise the ability that reproduction is weighed, the annual clinical interview that only need carry out limited number of times during the treatment.Treat and to cause that antigenicity problem, tumor form, teratoma forms or unusual neural the connection by giving hES cell and its derivant according to the present invention.

Simultaneously, giving (particularly SCI patient) hES cell and its derivant causes decubital ulcer to be cured, reduces demand, recovery bladder sensation and control, recovery intestinal sensation and pain and sense of touch to hypertension agents or antidepressants; Recover the reflection of disappearance; Alleviate the cold sweat symptom; Alleviate spinning sensation; The normalization of the normalization of blood pressure and the breathing that participates in by diaphragm fully.

Provide the method for the hES cell being suffered from the patient of subacute spinal cord injury or chronic spinal cord lesion and the failure of natural Restoration Mechanism below in detail.Also provide in detail below according to the present invention and damaged the distinct methods for the treatment of acute SCI as first-line treatment in three months.

Treatment effective dose, dosage regimen and the route of administration of the hES cell that preparation gives depends primarily on three kinds of factors: promptly disease type, patient's clinical condition and the seriousness of symptom takes place.In the invention process process, found to treat effective dose and scheme does not depend on age, sex, body weight or race.According to the independent scheme of setting up each patient of ongoing patient evaluation process.

In implementing an embodiment of the invention, the pharmaceutical composition that will contain hES cell and/or its derivant by a series of injections suffers from the disease that can't cure at present or the object of termination property disease or other clinical disease (as mentioned above), to treat this disease.

The various embodiments for the treatment of clinical disease and whole last disease according to the present invention are described below with reference to following examples.

Treatment, dosage, scheme, route of administration, assessment and SCI patient follow up a case by regular visits to

In one embodiment, by a series of injections hES cell and/or its derivant suffered from the object that SCI surpasses three months (subacute and chronic SCI), to treat this disease.In another embodiment, the hES derivant is the hemopoietic progenitor cell stem cell.In another embodiment, the hES derivant is the neuron progenitor cell stem cell.

Proof load

In one embodiment, detection is diluted to final volume with physiological saline solution and is about about 750 of 0.25-1.0ml, 000-80,000, the pollution of 000 hES cell and/or its derivant and vigor, and, being subcutaneously injected into forearm with administration as proof load then with the standard method counting, wherein said cell comprises hemopoietic progenitor cell stem cell and neuron progenitor cell stem cell.After 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour and 24 hours, observe to detect anaphylactic shock, injection site pain or inflammation, itch all over, blush or generate heat.In one embodiment, the ratio of hES cell and hemopoietic progenitor cell stem cell and neuron progenitor cell stem cell is about 4:1-1:4.In another embodiment, this ratio is about 1:1.

Initial dosage

Have an appointment 750 the containing of physiological saline solution that this scheme comprises is subcutaneous, intramuscular and/or the initial injection of intravenous are suspended in the about 0.25ml-1.0ml of volume, 000-80,000, the pharmaceutical composition of 000 hES cell and/or its derivant is with administration, and wherein said cell comprises hemopoietic progenitor cell stem cell and neuron progenitor cell stem cell.HES cell and its derivant of containing equal number by injection every day in 1 week-10 day are initially injected.

Epidural injection and epidural catheter injection

By initial injection after 7-10 days for three days on end in twice ground every day epidural injection or epidural catheter above or below the injury site inject.Be suspended in the physiological saline solution that volume is 2ml and with further dilution about 750 of 5ml-40ml physiological saline solution, 000-80,000,000 hES cell and/or its derivant, wherein said cell comprises the neuron progenitor cell stem cell.Clinical course and doctor's viewpoint according to the doing well,improving of patient performance repeat epidural injection/epidural catheter drug administration by injection above or below damage location.Also observe,, will significantly improve sensory nerve,, then significantly improve nervus motorius if prostrate if the patient lies on the back behind epidural injection.In both cases, all have to patient's lower limb is remained on the position of raising.

In the sheath

Initially injecting beginning 2,5,8,12,17 and being suspended in the 2ml physiological saline solution and further being diluted to cumulative volume with the 2ml physiological saline solution by intrathecal injection above or below damage location after 22 months is about 750 of 4ml, 000-11,000,000 hES cell and/or its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.

In one embodiment, for example, behind the treatment SCI, give cell by conduit epidural injection above or below the damage location back 15 days of damage.In one embodiment, injection is suspended in the 2ml volume physiological saline solution, is diluted to about 750 in the 15-40ml physiological saline solution then, 000-80,000,000 hES cell and/or its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.This treatment can repeat behind one and a half months.

Degree of depth spinal cord injection

The symptom reversal procedures that arrives according to the observation, can be suspended in about 750 in the 0.25-1.0ml volume physiological saline solution with containing, 000-80,000, the pharmaceutical composition of 000 hES cell and/or its derivant carries out booster injection once more, and wherein said cell comprises hematopoietic stem cell and neuron progenitor cell stem cell.In one embodiment, by weekly or degree of depth spinal cord injection week about give this pharmaceutical composition at the spinal column back side.This treatment can be strengthened muscle of back, in case the patient can activity after then can accelerate the physical rehabilitation.

The tail side injection

Put into practice according to the present invention, be resuspended in 2ml saline and be diluted to 10ml separately or by the tail side injection with about 750 of the dilution of 20ml DEPOMEDROL (acetic acid methylprednisolone) at the most, 000-80,000, the pharmaceutical composition of 000 hES cell and/or its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.This treatment can be strengthened lumbar region muscle, makes lumbar region and sacrum area regain sensation and motor capacity.

Except that the hES cell transplantation of studying, also use the motor function that loses and instruct to keep promoting that the healthy Lifestyle of cell function and cell regeneration process recovers the patient by daily physiotherapy, retraining patient.

Topical

Directly treat the SCI except that putting into practice according to the present invention, can fast and effeciently treat because the decubital ulcer that patient's extended immobilization inertia causes by the pharmaceutical composition that hES cell and/or its derivant are contained in the part with hES cell and/or its derivant.

Can contain by the part and have an appointment 750,000-160, the medicine composite for curing decubital ulcer of 000,000 hES cell and/or its derivant, wherein said cell comprises hemopoietic progenitor cell.Give loading dose or predose by intravenous and intramuscular injection, so that the inner formation of curing and promoting neovascularity.Perhaps, hES cell and its derivant are resuspended in the 2ml saline,, use in the damage with the dilution of 2-4ml saline.

Intravenous infusion

According to the present invention's practice, will contain 750,000-80, the pharmaceutical composition of 000,000 hES cell and/or its derivant is resuspended in the 100ml normal saline and by intravenous route and gives, and wherein said cell comprises hemopoietic and neuron progenitor cell.This treatment has guaranteed that stem cell continuously flows into human body, is particularly useful for because of certain reason, as decubital ulcer, the patient disabled situation of other direct route of administration that causes such as weakness too.

Treat the scheme of acute SCI

Can significantly improve patient's survival rate in the treatment of the acute SCI of intervention fast.And, by implement the present invention get involved fast any can not cure diseases, can improve the probability of fully recovering in the treatment of disease or imbalance.In yet another embodiment of the present invention, also can such as hereinafter detailed description, successfully treat and acute SCI take place after the spinal cord injury less than trimestral patient.According to the present invention's practice, the pharmaceutical composition of hES cell and/or its derivant and effective physiotherapy, recovery and retraining course are the effective gamma therapies of acute SCI.

Treat acute SCI and comprise two additional steps that add in steps that are used for the treatment of subacute and chronic SCI.When initial interference SCI takes place in damage after (as operation) immediately, directly give damage location with hES cell and/or its derivant.Successive treatment is included in twice intrathecal injection and epidural injection that damages in three months.

Clinical evaluation and observation

Lower limb are lost the patient of motor function long period, though can treat according to the present invention, should be with the recovery of its lower extremity motor function, walk retraining course and other physiotherapy.As first step in this course, proved that it is effective using walking frame and clamp in retraining.In addition, the muscle that should carry out upper limb and upper body strengthens course so that the patient can be in walking its body weight of independent support.Along with the carrying out of treatment and the improvement of patient's disease, can reduce the retraining course.

Except that the demand to medical apparatus and instruments support and physiotherapy reduces, because treat sympathetic symptom and the parasympathetic symptom of improving the patient by the present invention, so reduced the demand of patient, comprised fluctuation of blood pressure medicine, depressant drug, decubital ulcer medicine, the medicine that is used for intestinal and bladder complication and medical apparatus and instruments, anti--spasm medicine and pump to medicine.

Except that the treatment and the demand of the direct or indirect related indication medicine of SCI are reduced, observe also that the demand to antidiabetic drug also reduces when treating diabetes and SCI according to the present invention.

SCI to taking place and according to the clinical evaluation of the doing well,improving process that the patient showed of the present invention's treatment in routine monitoring in the therapeutic process and after the remission.Assess many physiologys, sympathetic nerve, parasympathetic nervous, nervus motorius, autonomic nerve and spiritual parameter,, describe in detail as follows to estimate the effectiveness that this technology reverses the SCI symptom.

Check the record of physician, comprise symptom description, damage location classification, symptom development, clinical intervention, treatment and damage medical history.Check and overhauling according to this kind the patient, comprise magnetic resonance imaging (MRI), electromyography (EMG), nerve conduction velocity (NCV) scanning and other neurologic examination and research (be included in treatment preceding assessment degree of injury and obtain the neurologic examination that damage is write down), carry out transplanting the method that back improvement carrying out sxemiquantitative is described, describe in detail as follows putting into practice according to the present invention.

Note sympathetic nerve parameter and neurological kilter, comprise patient's the mental status, whether depression, behavior characteristics, cranial nerve function and overall behavior, as the part of spirit evaluation.

Estimate sign and the symptom relevant with the autonomic nervous system function with the nerve injury of damage location.This comprises neurologic examination, detect ability, sense of touch, consciousness, the feels pain of perceived depth pressure ability, ability, involuntary movement that sensible temperature changes, be in a cold sweat, dizziness, blood pressure, dyspnea, abnormal posture and the ability that independently takes a seat when lying down.

Estimate bladder and function of intestinal canal.This comprises bladder control, bladder stream and feels that filling of bladder, intestinal control, intestinal drain time and intestinal sensation.

Estimate the upper body exercises function, with assessment central nervous system's degree of injury.This comprises head movement, wrist and finger motion, tendon reflex and quadruped locomotion intensity, amyotrophy and holds power.

Estimate lower part of the body motor function, with assessment central nervous system's degree of injury.This comprises buttocks motion, knee kinematics, toe movement, tendon reflex and quadruped locomotion intensity, amyotrophy and vola reaction.Also during treating, regularly carry out detailed neurologic examination, with the innerv progress of monitoring extremity.

In order to help the patient during clinical interview, to recover,,, and improve its motor capacity so that it recovers use extremity and joint along with functional recovery is regulated the patient with the physiotherapy technology of standard.

By the recovery of function of nervous system and the evidence of neurological evaluation demonstration neuranagenesis.The example of SCI therapeutic scheme is as case research in the present invention.

The nervous system development treatment of diseases

Give according to the present invention practice about 750,000-160,000,000 hES cell and/or its derivant are with treatment disorder of development such as autism and mental retardation.In another embodiment, give proof load after, give by intramuscular or intravenous route about 750,000-80,000,000 cell.Also can adopt intrathecal route.In another embodiment, also can adopt intracranial transplantation.HES cell and/or its derivant mainly are neuron progenitor cells.In another embodiment, give hemopoietic and neuron progenitor cell by intramuscular or intravenous route.This treatment is begun to continue 1 year by intramuscular injection every day, or continues 3 months through intravenous route.Then, carry out once identical injection weekly a middle of the month at ensuing 3-6, carry out once, carried out once in every month then in ensuing per two weeks in 3 middle of the month according to doctor's observation.Intrathecal injection only is included in the scheme after begin treatment 6-8 month, has only then and carry out intrathecal injection when the patient is reactionless to intramuscular and intravenous injection.In another embodiment of the present invention, with 750,000-80,000,000 hES cell and/or its derivant are resuspended in the 100ml normal saline, by the intravenous infusion administration.

Embodiment 1:

After containing the pharmaceutical composition of hES cell and its derivant, diagnosis suffers from that autism contacts with flapping tremor, hyperkinetic syndrome, no interpersonal skill, no sight line, rolling movement (pin rollingmovement), can not follow instruction, the patient that is unwilling to learn improves, described hES cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM with its derivant.

This patient's infusion protocol is referring to table 2.In relevant subsequently all forms of infusion protocol of this harmony in the exterior, the ＂ neuron ＂ in the cell type refers to promoting to be divided into the hES cell group who has partly broken up in the culture medium (as DMEM) of neural progenitor cell stem cell.This cell mass comprises it mainly being hES cell and the stem cell of neuron progenitor cell stem cell.Term ＂ non-neuron ＂ refers to promoting to be divided into the hES cell group who has partly broken up in the culture medium (as RPMI) of the CFU-GM stem cell except that the neuron progenitor cell stem cell.Cell mass comprises it not being hES cell and the various stem cell of neuron progenitor cell stem cell substantially.

Table 2

Date	Route of administration	Cell type
			3/13	Proof load	Non-neuron
3/14	im	Neuron
			3/16	im	Neuron
3/17	im	Neuron
			3/18	im	Neuron
3/20	im	Neuron
			3/21	im	Neuron
3/22	im	Neuron
			3/23	im	Non-neuron
3/24	im	Neuron
			3/27	im	Neuron
3/28	im	Non-neuron
			3/29	im	Non-neuron
3/30	im	Non-neuron
			3/31	im	Neuron
4/3	im	Neuron
			4/4	im	Neuron

4/5	im	Neuron
			4/6	im	Neuron
4/7	im	Neuron
			4/11	im	Neuron
4/12	im	Neuron
			4/13	im	Neuron
4/14	im	Neuron
			4/17	iv	Neuron
4/19	im	Neuron
			4/21	iv	Neuron
4/24	im	Neuron
			4/25	im	Neuron
4/28	iv	Neuron
			5/1	iv	Neuron
5/3	iv	Neuron
			5/5	im	Neuron
5/8	iv	Neuron
			5/10	im	Neuron
5/12	im	Neuron
			5/15	iv	Neuron
5/17	im	Neuron
			5/29	im	Neuron
5/31	iv	Neuron
			6/2	iv	Non-neuron
6/5	im	Neuron
			6/7	iv	Neuron
6/9	iv	Non-neuron
			6/12	iv	Neuron
6/14	The iv infusion	Neuron
			6/16	im	Neuron
6/19	im	Non-neuron
			6/21	im	Neuron
6/23	iv	Neuron
			7/3	iv	Neuron
7/5	The iv infusion	Neuron
			7/7	im	Non-neuron
7/10	im	Non-neuron
			7/12	im	Neuron
7/14	im	Neuron
			7/17	Iv infusion x 2	Neuron

7/18	im	Non-neuron
			7/20	im	Non-neuron
7/24	im	Non-neuron
			7/28	im	Non-neuron
7/31	im	Non-neuron
			8/2	im	Non-neuron
8/4	im	Non-neuron
			8/7	im	Non-neuron
8/10	im	Non-neuron
			8/11	im	Non-neuron
8/14	im	Non-neuron
			8/18	im	Non-neuron
8/21	im	Neuron
			8/23	im	Non-neuron
8/28	im	Non-neuron
			8/30	im	Non-neuron
9/1	im	Non-neuron
			9/4	im	Non-neuron

The nervous system degeneration treatment of diseases

Give about 750 according to the present invention's practice, 000-160,000,000 hES cell and/or its derivant, with treatment nervous system degeneration disease, include but not limited to: cortex-substrate degeneration, olivopontocerebellar atrophy, Alzheimer's disease, parkinson disease, multiple sclerosis, dementia, auditory nerve atrophy and motor neuron, wherein said cell comprise neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM.In another embodiment, give about 750,000-80,000,000 cell.

Though can change dosage regimen to be fit to concrete patient, but the typical scenario of treatment nervous system in children degenerative disease is included in intramuscular and intravenous injection every day first month, injected weekly three times at 2-4 month, the 5th and injection weekly in 6 months, 9-12 month booster injection weekly.In the adult, typical scenario comprises intramuscular and intravenous injection every day, and by in epidural catheter, lumbar puncture, the sheath or tail side administration.Afterwards, carry out intravenous infusion twice, at least at interval 7 days therebetween.At last, give booster injection 6 every month middle of the month, after per 2 months of at least 6 middle of the month be administered once.Because the carrying out property of disease continues the treatment long period, even may last for life.

Under the situation of carrying out property and degenerative disease, stop or the stable disease process can improve self dependency of patient by getting involved fast according to the present invention.After taking place, Stabilization promptly can be observed some improvement.

Embodiment 2:

The patient who takes SOLUMEDROL (methylprednisolone) every day who suffers from 2 years carrying out property of repeatability multiple sclerosiss can't walk, can not control bladder or intestinal, and the frequent respiratory tract disease that takes place.

According to the present invention's treatment, the object disease improves, and the patient can walk, and recovers the control to bladder and intestinal, and the consumption of SOLUMEDROL is reduced.MRI demonstrates 50% to be improved.

This patient's infusion protocol is referring to table 3.

Table 3

Date	Route of administration	Cell type
			7/15	Proof load	Neuron
7/25	im	Neuron
			7/26	im	Neuron
7/27	im	Non-neuron
			7/28	im	Neuron
7/29	im	Neuron
			8/1	im	Neuron
8/2	im	Neuron
			8/3	im	Neuron
8/4	im x 2	Neuron
			8/5	im x 2	Neuron
8/9	im x 2	Neuron
			8/10	im x 2	Neuron
8/11	im	Neuron
			8/12	im x 2	Neuron
8/16	iv im	Neuron
			8/22	im x 4	hES
8/24	iv im	Neuron
			9/2	iv	Neuron
9/8	im	Neuron
			9/15	im	Neuron and non-neuron mixture
9/19	iv	Neuron
			9/22	im	Neuron
9/26	im	Neuron and non-neuron mixture
			9/29	im	Neuron and non-neuron mixture

10/3	im	Neuron and non-neuron mixture
			10/6	im	Non-neuron
10/13	iv	Neuron and non-neuron mixture
			10/17	im	Neuron and non-neuron mixture
10/20	iv	Neuron
			10/24	iv	Neuron and non-neuron mixture
10/31	iv	Neuron and non-neuron mixture
			11/3	iv im	Neuron and non-neuron mixture
11/7	im	Non-neuron
			11/21	iv	Non-neuron
11/24	im	Non-neuron
			1/19	im x 2	The non-neuron neuron
1/25	im x 2	Neuron
			2/2	im iv	The non-neuron neuron
2/15	im x 2 iv	The neuron non-neuron
			2/23	im x 2 iv	The neuron non-neuron
3/2	im x 2 iv	The neuron non-neuron
			3/8	im x 2 iv	Non-neuron
3/16	im x 2 iv	The neuron non-neuron
			5/2	im x 2	Non-neuron
5/12	im x 2 iv	The neuron non-neuron
			6/8	im x 2 iv	The neuron non-neuron
6/16	im x 2 iv	The neuron non-neuron
			7/3	im	Neuron
7/4	im	Neuron
			7/5	im	Neuron
7/6	im	Neuron
			7/7	im	Neuron
7/18	im x 2 iv	Neuron

		Non-neuron
			7/22	im x 2 iv	The neuron non-neuron
7/24	im x 2 iv	The neuron non-neuron
			8/1	im x 2 iv	Non-neuron
8/4	im x 2 iv	Non-neuron
			8/7	im x 2 iv	Non-neuron
8/24	im x 2 iv	Non-neuron
			8/28	im x 2 iv	Non-neuron
9/2	im x 2 iv	Non-neuron
			9/6	im x 2 iv	Non-neuron
9/9	im x 2 iv	Non-neuron

Embodiment 3:

ALS (motor neuron) took place in 60 years old male before 2 years, its upper limb and hands worsen rapidly.His upper limb, hands and tongue continue to take place ballism and fasciculation.Can not rotate left arm up and down, or left arm be lifted high excessive.Right arm can run and lifted, and hands is the skeleton shape.

After treating a year and a half, by the regeneration of both hands hypothenar and palm muscle, his hands recovers from skeleton shape framework.Both arms can both rotate up and down.He finds to have equally dysphagia, but does not worsen from treatment.His lower limb, breathing and linguistic competence are not worsened.

This patient's infusion protocol is referring to table 4.

Table 4

Date	Route of administration	Cell type
			8/2	im	Neuron (proof load)
8/4	im	The neuron non-neuron
			8/5	im	Neuron
8/8	im	Neuron
			8/9	im	Neuron

8/10	im	Neuron
			8/11	im	Neuron
8/12	im	Neuron
			8/16	im	Neuron
8/17	im	hES
			8/22	im	Neuron
8/23	im	Neuron
			8/24	im x 3	Non-neuron
8/25	im x 3	Non-neuron
			8/26	im	Neuron
8/29	im	Neuron
			8/31	im iv	Neuron
9/2	im	Neuron
			9/5	im iv	Neuron
9/7	im	Neuron
			9/9	im iv	Neuron mixes
9/12	im iv	The neuron non-neuron
			9/13	im iv	The neuron non-neuron
9/16	im	Mix
			9/19	im iv	Neuron mixes
9/22	iv	Neuron
			9/23	im iv	The neuron non-neuron
9/26	im	Mix
			9/27	im	Mix
9/28	im iv	The neuron non-neuron
			9/30	im iv	Mix non-neuron
10/3	iv	Mix
			10/7	im	Non-neuron
10/10	iv	Neuron
			10/12	iv	Mix
10/17	im iv	Neuron

		Mix
			10/20	Epidural	Neuron
10/21	iv	Neuron
			10/24	iv	Mix
10/27	iv	Neuron
			10/28	iv	Mix
10/31	iv	Mix
			11/2	iv	Mix
11/3	Epidural	Neuron
			11/4	iv	Mix
11/7	im	Non-neuron
			11/9	im	Non-neuron
11/11	im	Neuron
			11/14	im	Neuron
11/17	im	Non-neuron
			11/21	im	Non-neuron
11/23	im	Non-neuron
			12/13	im	Non-neuron
12/19	im	Neuron
			12/21	im	Neuron
12/23	im	Neuron
			12/29	im	Neuron
1/3	im	Neuron
			1/9	im	Neuron
1/12	im	Neuron
			1/13	iv	Non-neuron
1/16	iv	Neuron
			1/19	im iv	The neuron non-neuron
1/23	Epidural	Neuron
			1/27	im iv	The neuron non-neuron
1/29	im iv	The neuron non-neuron
			2/1	iv	Neuron
2/3	im iv	The neuron non-neuron
			2/6	im iv	The neuron non-neuron
2/8	iv	Non-neuron

2/10	iv	Non-neuron
			2/13	iv	Non-neuron
2/15	iv	Non-neuron
			2/17	im iv	The neuron non-neuron
2/20	im iv	The neuron non-neuron
			2/22	im iv	The neuron non-neuron
2/24	im iv	The neuron non-neuron
			2/27	The iv mouthspray	The neuron non-neuron
3/1	im iv	The neuron non-neuron
			3/6	iv	Non-neuron
3/8	Epidural	Neuron
			3/9	Epidural	Neuron
3/10	Epidural	Neuron
			3/11	iv	Neuron
3/22	im	Non-neuron
			3/24	im	Non-neuron
3/27	iv	Non-neuron
			3/29	im iv	Non-neuron
3/31	The im mouthspray	The neuron non-neuron
			4/3	im iv	The neuron non-neuron
4/5	im iv	The neuron non-neuron
			4/7	im iv	The neuron non-neuron
4/10	im iv	The neuron non-neuron
			4/12	im iv	The neuron non-neuron
4/19	Epidural	Neuron
			4/24	iv	Neuron
4/26	iv	Non-neuron

4/28	im iv	The neuron non-neuron
			5/2	im	Non-neuron
5/3	im	Neuron
			5/5	im iv	The neuron non-neuron
5/8	iv	Non-neuron
			5/11	Intravenous infusion	Neuron
5/12	im	Non-neuron
			5/14	im iv	The neuron non-neuron
5/15	im iv	The neuron non-neuron
			5/16	im iv	The neuron non-neuron
5/17	im iv	The neuron non-neuron
			5/19	im iv	The neuron non-neuron
5/22	im iv	The neuron non-neuron
			5/24	im iv	The neuron non-neuron
5/26	im iv	The neuron non-neuron
			5/29	im iv	The neuron non-neuron
6/1	im iv	The neuron non-neuron
			6/2	im iv	The neuron non-neuron
6/5	Epidural catheter	Neuron
			6/6	Epidural catheter	Neuron
6/7	Epidural catheter	Neuron
			6/12	im	The neuron non-neuron
6/14	im iv	The neuron non-neuron
			6/16	Intravenous infusion	Neuron
6/21	im	Neuron

6/24	im	Neuron
			7/3	iv	Neuron
7/5	iv	Neuron
			7/7	iv	Neuron
7/10	iv	Neuron
			7/12	iv	Neuron
7/14	iv	Neuron
			7/17	iv	Non-neuron
7/20	iv	Non-neuron
			7/24	iv	Non-neuron
7/25	Epidural (in the sheath)	Non-neuron
			7/31	iv	Neuron
8/2	iv	Neuron
			8/3	iv	Non-neuron
8/8	iv	Non-neuron
			8/11	iv	Non-neuron
8/16	iv	Neuron
			8/23	iv	Neuron
8/28	iv	Non-neuron
			8/29	iv	Non-neuron
9/5	iv	Non-neuron
			9/6	iv	Non-neuron
9/8	iv	Non-neuron
			9/13	iv	Non-neuron
9/15	im iv	Non-neuron
			9/18	im iv	Non-neuron
9/19	Intravenous infusion	Neuron
			9/20	Intravenous infusion	Non-neuron
9/22	im iv	Non-neuron
			9/25	im iv	Non-neuron
9/27	im iv	Non-neuron
			9/29	im iv	Non-neuron
10/2	im iv	Non-neuron
			10/4	im iv	Non-neuron

10/9	im iv	Non-neuron
			10/13	im iv	Non-neuron
10/16	im iv	Non-neuron
			10/18	im iv	Non-neuron
10/20	im iv	Non-neuron
			10/23	im iv	Non-neuron
10/25	The iv infusion	Mix
			10/27	The iv infusion	Non-neuron
11/1	im iv	Non-neuron
			11/6	im iv	Non-neuron
11/8	im iv	Non-neuron
			11/10	In the sheath	Neuron
11/13	im iv	Non-neuron
			11/15	im iv	Non-neuron
11/17	im iv	The neuron non-neuron
			11/20	im iv	Non-neuron
11/22	im iv	Non-neuron
			11/24	iv	Neuron
11/27	im iv	Mix
			12/1	im iv	Mix
12/4	The iv infusion	Mix
			12/6	im iv	Non-neuron
12/8	iv	Neuron
			12/11	im iv	Non-neuron
12/14	The iv infusion	Non-neuron
			12/15	The iv infusion	Non-neuron

12/18	im iv	Non-neuron
			12/20	iv	Neuron
12/27	im iv	Neuron
			12/29	im iv	Neuron
1/4	im iv	Non-neuron
			1/8	The iv infusion	Mix
1/9	The iv infusion	Mix
			1/10	The iv infusion	Mix
1/12	im iv	Non-neuron
			1/16	im iv	Non-neuron
1/19	im iv	Non-neuron
			1/22	im iv	Mix

Embodiment 4:

The patient is suffered from parkinson disease by diagnosis.The patient is reactionless to standard care, and the state of an illness worsens in time.He has typical shuffling gait.One-sided trembling appears in right arm, and he makes people worried on physiology very much.He can't open eyes.After the treatment beginning, this patient is progressively improved, and behind 1 year hES cell therapy, this patient significantly improves.Its gait is normal, and trembling of hands alleviates, mental status optimism.He can open eyes fully.And begin to sign and write, this is impossible when the treatment beginning.At present very little to other people dependency.

This patient's infusion protocol is referring to table 5.

Table 5

Date	Route of administration	Cell type
			1/30	im iv	Neuron (proof load)
1/31	im	Neuron
			2/1	iv	Neuron
2/2	iv im	The non-neuron neuron
			2/3	iv im	Neuron

2/4	iv im	Neuron
			2/5	iv im	Neuron
2/6	iv im	The non-neuron neuron
			2/8	iv im	The non-neuron neuron
2/9	iv im	The non-neuron neuron
			2/10	iv im	The non-neuron neuron
2/11	iv im	Non-neuron
			2/12	iv im	The neuron non-neuron
2/13	iv im	The neuron non-neuron
			2/14	iv	Neuron
2/15	iv im	The neuron non-neuron
			2/16	iv im	The neuron non-neuron
2/17	iv im	The neuron non-neuron
			2/18	iv im	The neuron non-neuron
2/19	iv im	The neuron non-neuron
			2/20	iv im	The neuron non-neuron
2/21	iv im	The neuron non-neuron
			2/23	iv im	The neuron non-neuron
2/25	iv im	The neuron non-neuron
			2/26	iv im	The neuron non-neuron
2/27	iv im	The neuron non-neuron

2/28	iv	Neuron
			3/1	iv im	The neuron non-neuron
3/2	iv im	The neuron non-neuron
			3/3	iv	Neuron
3/4	im	Neuron
			3/5	iv	Neuron
3/7	im	Neuron
			3/9	iv	Non-neuron
3/10	iv im	The neuron non-neuron
			3/11	iv im	The neuron non-neuron
3/12	iv im	The neuron non-neuron
			3/13	iv im	The neuron non-neuron
3/28	iv	Neuron
			3/29	iv im iv im	The neuron non-neuron
3/30	iv im im	The neuron non-neuron
			3/31	iv im	The neuron non-neuron
4/1	iv im	The neuron non-neuron
			4/2	iv im	The neuron non-neuron
4/3	iv im	The neuron non-neuron
			4/4	iv im im	The neuron non-neuron
4/5	iv im	The neuron non-neuron
			4/6	iv im	The neuron non-neuron
4/7	iv im im	The neuron non-neuron
			4/8	iv	Neuron

	im	Non-neuron
			4/9	iv im	The neuron non-neuron
4/10	iv im	The neuron non-neuron
			4/11	iv im	The neuron non-neuron
4/12	iv im	The neuron non-neuron
			4/13	iv im	The neuron non-neuron
4/14	iv im	The neuron non-neuron
			4/15	iv im	The neuron non-neuron
4/16	iv im	The neuron non-neuron
			5/20	iv im	The neuron non-neuron
5/21	Infusion	Non-neuron
			7/10	iv im	The neuron non-neuron
7/11	iv im	The neuron non-neuron
			7/12	iv im	The neuron non-neuron
7/13	iv im	The neuron non-neuron
			7/14	iv im	The neuron non-neuron
7/15	The iv infusion	Non-neuron
			7/16	The iv infusion	Non-neuron
7/17	iv im	The neuron non-neuron
			7/18	iv im	The neuron non-neuron
7/19	iv im	The neuron non-neuron
			7/20	iv im	The neuron non-neuron

7/21	iv im	The neuron non-neuron
			7/22	The iv infusion	Neuron
7/23	The iv infusion	Neuron
			7/24	The iv infusion	Non-neuron
7/25	iv im	The neuron non-neuron
			7/26	iv im	The neuron non-neuron
7/27	iv im	The neuron non-neuron
			7/28	iv im	Non-neuron
7/29	The iv infusion	Neuron
			7/30	im	The neuron non-neuron
9/9	im iv	Non-neuron
			9/12	im iv	Non-neuron
9/13	iv im	Non-neuron
			9/14	im iv	Non-neuron
9/15	im iv	Non-neuron
			9/16	im iv	Non-neuron
9/17	The iv infusion	Neuron
			9/18	The iv infusion	Neuron
9/19	iv	Neuron
			9/20	im iv	Non-neuron
9/21	im iv	Non-neuron
			9/22	im iv	Non-neuron
9/23	The iv infusion	Non-neuron
			9/24	The iv infusion	Non-neuron
9/25	iv im	Non-neuron
			9/26	iv im	Non-neuron

9/27	iv im	Non-neuron
			9/28	iv im	Non-neuron
9/29	Iv iv infusion	Neuron
			9/30	iv im	Non-neuron
11/9	iv im	Non-neuron
			11/10	iv im	Non-neuron
11/11	iv im	Non-neuron
			11/12	iv im	Non-neuron
11/14	The iv infusion	Mix
			11/16	iv im	Non-neuron
11/17	iv im	Non-neuron
			11/18	The iv infusion	Neuron
11/19	The iv infusion	Neuron
			11/20	iv im	Non-neuron
11/21	iv im	Non-neuron
			11/22	iv im	Non-neuron
11/23	iv im	Non-neuron
			11/24	The iv infusion	Neuron
11/25	The iv infusion	Neuron
			11/26	im iv	Mix
11/29	iv im	Non-neuron
			1/9	iv im	Non-neuron
1/10	iv im	Non-neuron
			1/11	iv im	Non-neuron
1/12	iv im	Non-neuron

1/13	The iv infusion	Mix
			1/14	The iv infusion	Mix
1/15	iv im	Non-neuron
			1/16	iv im	Non-neuron
1/17	iv im	Non-neuron
			1/18	iv	Non-neuron
1/19	iv	Non-neuron
			1/20	The iv infusion	Non-neuron
1/21	The iv infusion	Mix
			1/22	im iv	Mix
1/23	im iv	Mix
			1/24	iv im	Neuron
1/25	iv im	Neuron
			1/26	iv im	Neuron
1/27	The iv infusion	Mix
			1/28	The iv infusion	Mix
1/29	iv im	Neuron
			1/30	iv im	Neuron

Cerebral palsy's treatment

Give according to the present invention practice about 750,000-160,000,000 hES cell and/or its derivant, with the treatment cerebral palsy, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM.This scheme is identical with the treatment of neurodegenerative disease with dosage.

Embodiment 5:

48 years old male that when birth is diagnosed as CP at AIIMS has the symptom of slurred speech, shuffling gait, dysphagia and left side hemiplegia.The chart of drawing muscle tonus, reflection and strength is to confirm these symptoms.

Treat after 6 months, that his speech becomes is clear, absent deglutition is difficult, can use left side body and gait to improve.The enough left hands of energy shave and can pick up teacup etc. by enough left hands.His balanced capacity is better, can walk for a long time.

This patient's infusion protocol is referring to table 6.

Table 6

Date	Route of administration	Cell type
			3/8	Proof load	Non-neuron
3/10	iv im	Neuron
			3/17	iv im	Neuron
4/8	iv im x 2	Neuron
			4/9	iv x 3 im x 3	The neuron non-neuron
4/10	iv x 3 im x 3	The neuron non-neuron
			5/10	iv im	The neuron non-neuron
5/11	im x 2 iv	Neuron
			5/12	Iv infusion x 2	Neuron
5/13	The iv infusion	Non-neuron
			5/14	Iv infusion iv im	The neuron non-neuron
5/15	The iv infusion	Non-neuron
			7/19	iv im	The neuron non-neuron
7/20	The iv infusion	Non-neuron
			7/21	Iv infusion x 2	Non-neuron
7/22	iv im	The neuron non-neuron
			7/24	iv im	The neuron non-neuron
7/25	Degree of depth spinal cord injection	Neuron
			7/26	iv im	The neuron non-neuron
7/27	iv im	The neuron non-neuron
			7/28	iv	Non-neuron

	im
			7/31	iv im	The neuron non-neuron
8/1	iv im	Non-neuron
			8/2	iv im	Non-neuron
8/3	iv im	Non-neuron
			8/4	Iv infusion x 2	Non-neuron
8/5	The iv infusion	Non-neuron
			8/7	iv im	Non-neuron
8/8	iv im	Non-neuron
			8/9	iv im	Non-neuron
8/10	iv im	Non-neuron
			8/11	iv im	Non-neuron
8/12	iv im	Non-neuron
			8/14	Degree of depth spinal cord injection	Neuron
8/16	The iv infusion	Neuron
			8/17	The iv infusion	Neuron
8/18	iv im	Neuron
			8/19	Degree of depth spinal cord injection	Neuron
8/20	iv im	Non-neuron
			8/21	iv im	Non-neuron
8/22	iv im	Non-neuron
			8/24	The iv infusion	Non-neuron
8/25	The iv infusion	Non-neuron
			8/26	iv im	Non-neuron

Embodiment 6:

The 3 years old girl who suffers from CP brought into the clinic, similar one month big baby of her outer harmony in the exterior behavior, uncontrollable cervical region, weak cry, reactionless and can not suck feeding bottle.She definitely is loose unable.

After treatment a year and a half, she becomes to look and looks and look like 2 years old baby.Have the cervical region control force, can recognize father and mother, the normal diet of creeping in bed, take food, smile when recognizing and take a seat having under the condition of support, can walk several steps when her mother embraces her.Her gait is a scissor gait, and she also begins to call its father and mother.

This patient's infusion protocol is referring to table 7.

Table 7

Date	Route of administration	Cell type
			8/4	im	Neuron (proof load)
8/5	im	Neuron
			8/8	im	Neuron
8/9	im	Neuron
			8/10	im	Neuron
8/11	im	Neuron
			8/12	im	Neuron
8/16	im	Neuron
			8/17	im	Neuron
8/18	im	Neuron
			8/19	im	Non-neuron
8/22	im	Mix
			8/23	im	Mix
8/25	im	Mix
			8/26	im	Neuron
8/29	im	Neuron
			8/30	im	Neuron
8/31	im	Neuron
			9/1	im	Neuron
9/2	im	Neuron
			9/5	im	Non-neuron
9/6	im	Neuron
			9/7	im	Neuron
9/8	im	Neuron
			9/12	im	Mix
9/14	im	Non-neuron
			9/16	im	Mix
9/19	im	Neuron

9/23	im	Neuron
			9/26	im	Mix
9/28	im	Mix
			9/30	im	Mix
10/3	im	Mix
			10/5	im	Neuron
10/10	im	Non-neuron
			10/17	im	Mix
10/19	im	Non-neuron
			10/21	im	Neuron
10/24	im	Mix
			10/28	im	Mix
10/31	im	Mix
			11/4	im	Mix
11/7	im	Non-neuron
			11/9	im	Non-neuron
11/11	im	Neuron
			11/14	im	Neuron
11/15	im	Non-neuron
			11/16	im	Non-neuron
11/18	im	Non-neuron
			11/22	im	Non-neuron
11/23	im	Non-neuron
			11/25	m	Non-neuron
11/28	im	Mix
			11/29	im	Mix
12/2	im	Mix
			12/7	im	Non-neuron
12/9	m	Non-neuron
			12/12	im	Non-neuron
12/14	m	Neuron
			12/16	im	Neuron
12/19	im	Non-neuron
			12/21	m	Neuron
12/23	im	Neuron
			12/26	m	Non-neuron
12/30	im	Neuron
			1/5	im	Neuron
1/11	im	Neuron
			1/13	im	Neuron

1/16	im	Neuron
			1/18	im	Neuron
1/23	im	Neuron
			1/25	im	Neuron
1/27	m	Neuron
			1/30	im	Neuron
2/1	im	Neuron
			2/3	im	Neuron
2/6	im	Neuron
			2/8	im	Neuron
2/10	im	Neuron
			2/13	im	Neuron
2/15	im	Neuron
			2/17	im	Non-neuron
22.2.06	im	Neuron
			2/24	im	Neuron
2/27	im	Neuron
			3/1	m	Neuron
3/3	im	Neuron
			3/6	im	Non-neuron
3/8	im	Non-neuron
			3/22	im	Non-neuron
3/24	im	Neuron
			3/27	im	Non-neuron
3/29	im	Neuron
			3/31	im	Neuron
4/7	im	Neuron
			4/10	im	Neuron
4/12	im	Neuron
			4/14	im	Neuron
4/17	im	Neuron
			4/19	im	Neuron
4/21	im	Neuron
			4/24	im	Neuron
4/26	im	Neuron
			5/1	im	Neuron
5/8	im	Non-neuron
			5/18	im	Neuron
5/22	im	Neuron
			5/26	im	Neuron

5/29	im	Neuron
			5/31	im	Neuron
6/2	im	Neuron
			6/5	im	Neuron
6/12	im	Neuron
			6/16	im	Neuron
6/19	im	Neuron
			6/21	im	Neuron
6/28	im	Neuron
			6/30	im	Neuron
7/4	im	Neuron
			7/7	im	Neuron
7/17	im	Neuron
			7/19	m	Neuron
7/21	im	Neuron
			7/24	im	Neuron
7/26	im	Neuron
			7/31	im	Non-neuron
8/2	im	Non-neuron
			8/4	im	Non-neuron
8/7	im	Non-neuron
			8/9	im	Non-neuron
8/11	im	Non-neuron
			8/14	im	Non-neuron
8/16	im	Non-neuron
			8/18	im	Non-neuron
8/21	im	Non-neuron
			8/23	im	Non-neuron
8/25	im	Non-neuron
			8/28	im	Non-neuron
8/30	im	Non-neuron
			9/1	im	Non-neuron
9/4	im	Non-neuron
			9/11	im	Non-neuron
9/13	im	Non-neuron
			9/15	im	Non-neuron
9/20	im	Non-neuron
			9/25	im	Non-neuron
9/27	im	Non-neuron
			10/3	im	Non-neuron

10/4	im	Non-neuron
			10/13	im	Non-neuron
10/16	im	Non-neuron
			10/18	im	Non-neuron
10/19	im	Non-neuron
			10/23	im	Non-neuron
10/25	im	Non-neuron
			10/27	im	Non-neuron
10/30	im	Non-neuron
			10/31	im	Non-neuron
11/3	im	Non-neuron
			11/6	im	Non-neuron
11/8	im	Non-neuron
			11/10	im	Non-neuron
11/13	im	Non-neuron
			11/15	im	Non-neuron
11/17	im	Non-neuron
			11/20	im	Non-neuron
11/22	im	Non-neuron
			11/24	im	Non-neuron
11/27	im	Non-neuron
			11/29	im	Non-neuron
12/1	im	Non-neuron
			12/8	im	Non-neuron
12/11	im	Non-neuron
			12/15	im	Non-neuron
12/26	im	Neuron
			12/27	im	Neuron
1/3	im	Non-neuron
			1/7	im	Non-neuron
1/15	im	Neuron
			1/24	im	Neuron
1/25	im	Neuron
			1/29	im	Neuron

Embodiment 8:

The child was suffered from cerebral palsy and serious mental retardation by diagnosis in 8 years old, can't independently walk.She can not recognition object, and span of attention is very low.Frequent sialorrhea, and always opening face.

After the treatment, she is sialorrhea no longer, and its span of attention improves, and looks cleverer.Can under situation about seldom supporting, walk.

This patient's infusion protocol is referring to table 8.

Table 8

Date	Route of administration	Cell type
			10/9	im	Non-neuron (proof load)
10/10	im	Non-neuron
			10/11	im	Non-neuron x2
10/12	im	Non-neuron
			10/13	im	Non-neuron
12/5	im	Non-neuron
			12/6	im iv	Non-neuron
12/7	im x 3	Non-neuron
			12/8	im iv	Non-neuron
12/9	im iv	Non-neuron
			12/10	im x 3	Non-neuron
12/11	im iv	Non-neuron
			12/12	im x 3	Non-neuron
12/13	im iv	Non-neuron
			12/14	im	Non-neuron
12/15	im iv	Non-neuron
			12/16	im x 3	Non-neuron
12/17	im iv	Non-neuron
			12/18	im	Non-neuron
12/19	im iv	Non-neuron
			12/20	im x 3	Non-neuron
12/21	im iv	Non-neuron
			12/22	im iv	Non-neuron
12/23	im iv	Non-neuron
			12/24	im	Non-neuron

	iv	Neuron
			12/25	im iv	Neuron
12/26	im iv	Neuron mixes
			12/27	im x 3	Neuron
12/28	im iv	Neuron
			12/29	im x 3	Neuron
12/30	im iv	Neuron
			12/31	im x 3	Neuron
1/1	im iv	Mix
			1/9	The iv infusion	Mix
1/10	The iv infusion	Mix
			1/11	im iv	Non-neuron
1/12	im iv	Non-neuron
			1/13	im iv	Non-neuron
1/14	im iv	Non-neuron
			1/15	im x 3	Non-neuron
1/16	im iv	Non-neuron
			1/17	im x 3	Non-neuron
1/18	im	Non-neuron
			1/19	im	Non-neuron
1/20	im	Non-neuron
			1/21	im	Mix
1/22	im	Mix
			1/23	im	Mix
1/24	im	Neuron
			1/25	im	Neuron
1/26	im	Mix
			1/27	im	Mix
1/28	im iv	Neuron
			1/29	im iv	Neuron

1/30	im iv	Neuron
			1/31	im	Non-neuron
1/2	iv	Non-neuron
			2/2	im	Non-neuron

The treatment of nervous system injury

According to the present invention practice, give about 750,000-160,000,000 hES cell and/or its derivant are with the treatment nervous system injury, include but not limited to brain injury, stupor and vegetative state, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM.In another embodiment, give about 750,000-80,000,000 cell.

Though can change dosage regimen being fit to concrete patient, the typical scenario of treatment nervous system injury be included in preceding 2 months every days intramuscular and intravenous injection and sheath in and epidural injection.

The treatment of cerebrovascular accident or apoplexy

According to the present invention practice, give about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM, with treatment cerebrovascular accident or apoplexy.In another embodiment, give about 750,000-80,000,000 cell.

Though can change dosage regimen to be fit to concrete patient, but the typical scenario of treatment cerebrovascular accident is included in intramuscular and two weeks of intravenous injection in first in middle of the month, intravenous infusion is 3 days then, at intravenous infusion 2 days in per 15 days of the 2nd and 3 middle of the month, at 5th month intravenous infusion 2 days and intrathecal injection, the 8th, 10 and 12 months intravenous infusions 4 days intramuscular injection 4 days then.

Embodiment 9:

The symptom that diagnosis suffers from the patient right side hemiplegia of cerebrovascular accident or apoplexy and has sialorrhea, dysphagia and can not talk and walk.In addition, this patient suffers from colon cancer.After the present invention's treatment, along with the raising of speech and motor activity, this patient demonstrates and improves sign, and spinal column becomes straight and hemiplegia is cured.

This patient's infusion protocol is referring to table 9.

Table 9

Date	Route of administration	Cell type
			8/17	Proof load	Non-neuron
8/22	im	hES
			8/23	im x 2	Neuron hES
8/24	im x 2	hES
			8/25	im	hES
8/26	im x 2	Neuron
			8/29	im	Neuron
8/30	iv	Neuron
			8/31	im	Neuron
9/2	im x 2	Neuron
			9/5	iv	Non-neuron
9/6	iv	Neuron
			9/7	im	Neuron
9/8	iv	Neuron
			9/9	iv	Neuron
9/12	im	Neuron and non-neuron mixture
			9/13	iv	Neuron
9/14	im	Non-neuron
			9/19	iv	Neuron
9/22	iv	Neuron
			9/26	im	Neuron and non-neuron mixture
9/29	iv	Neuron
			10/3	im	Neuron and non-neuron mixture
10/6	im	Non-neuron
			10/10	iv	Neuron
10/13	iv	Neuron
			10/17	iv	Neuron and non-neuron mixture
10/20	iv	Non-neuron
			10/24	iv	Neuron and non-neuron mixture
10/27	iv	Neuron
			10/31	iv	Neuron and non-neuron mixture
11/3	iv	Neuron and non-neuron mixture
			11/7	im	Non-neuron
11/10	iv	Non-neuron
			11/14	im	Neuron
11/17	im	Non-neuron
			11/21	im	Non-neuron

11/24	im	Non-neuron
			12/4	iv	Neuron and non-neuron mixture
12/12	iv	Non-neuron
			12/19	iv	Neuron
12/26	iv	Non-neuron
			1/4	iv	Neuron
1/11	The iv infusion	Neuron
			8/18	iv im	Neuron
8/21	iv im	Non-neuron
			8/22	iv im	Non-neuron
8/24	im iv	Non-neuron
			9/1	The iv infusion	Non-neuron
9/2	The iv infusion	Non-neuron

Embodiment 9:

Apoplexy took place in the male before 5 years in 82 years old, the right side hemiplegia occurred, and facial asymmetric and slurred speech.Use crutch to walk limping, because of being difficult to coordinate and to have a meal with the right hand.He can not independently take a seat or get up, and is pulling step when walking.Sialorrhea, and can not swallow well.

Treatment after two months, he can walk under unsupported situation, independently takes a seat and stands, and has a meal with the right hand, does not have slurred speech, does not have facial asymmetric.He can stand up from chair.He can be in walking crooked ill knee and keep balance.

This patient's infusion protocol is referring to table 10.

Table 10

Date	Route of administration	Cell type
			11/4	im	Non-neuron (proof load)
11/6	im	Non-neuron
			11/7	im	Neuron
11/8	im	Non-neuron
			11/9	im	Neuron
11/10	im	Neuron
			11/11	im	Non-neuron
11/13	im	Non-neuron
			11/14	im	Non-neuron

11/15	iv im	Neuron
			11/16	iv im	Neuron
11/18	iv im	Non-neuron
			11/22	The iv infusion	Neuron
11/23	The iv infusion	Neuron
			11/24	im	Neuron
11/27	im	Neuron
			11/28	im	Non-neuron
11/29	im	Neuron
			11/30	im	Neuron
12/2	im	Non-neuron
			12/4	im	Non-neuron
12/5	The iv infusion	Neuron
			12/6	Iv infusion im	Mix
12/7	im	Mix
			12/8	im	Mix
12/9	im	Mix
			12/11	im	Mix
12/12	im	Mix
			12/14	im iv	Mix
12/15	im	Mix
			12/16	im	Mix
12/18	im	Mix
			12/19	im iv	Mix
12/20	im	Mix
			12/21	im	Mix
12/22	im	Mix
			12/23	im	Mix
12/25	im	Neuron
			12/26	im	Neuron
12/27	iv	Mix
			12/28	im	Neuron
12/29	im	Neuron
			12/30	im	Neuron
1/1	im	Mix

1/2	im	Mix
			1/3	iv	Non-neuron
1/4	im	Non-neuron

The treatment of familial nervous system disease

Put into practice according to the present invention, with about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises that neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM give object, with treatment familial nervous system disease, includes but not limited to: olivary body pons brain atrophy and Huntington chorea.In another embodiment, give about 750,000-80,000,000 cell.Route of administration is in intramuscular and intravenous and the sheath, epidural catheter and intravenous infusion.This treatment will continue throughout one's life probably, but is high-intensity therapeutic in 1 year, subsequently, carries out intramuscular and intravenous injection at least 3 every days in the middle of the month, also gives in the sheath therebetween, epidural catheter and intravenous infusion.Behind the one and a half months of interval, gave same group of injection in 21 days.Continue every day and carry out intramuscular and intravenous injection, be reduced to then on every Wendesdays time, twice weekly, once in a week, whenever biweekly, once this depended on patient's situation in every month then.Can repeat intrathecal injection and epidural catheter in individual month interval of 4-6.Per two weeks, every month or per two months give intravenous infusion one time.

Embodiment 10:

Diagnosis suffers from sporadic spinocerebellar ataxia, can't change in the time of can not going to bed, walk or take a seat the position patient's dyslalia, tremble and can not lift head and neck.

After carrying out stem-cell therapy according to the present invention practice, notice that all symptoms all improve, this patient can balance himself also walk several steps.It is coherent that speech becomes.

This patient's infusion protocol is referring to table 11.

Table 11

Date	Route of administration	Cell type
			8/8	Proof load	Non-neuron
8/9	im	Neuron
			8/10	im x 2	Neuron
8/11	im x 2	Neuron
			8/12	im x 2	Neuron
8/13	im x 2	Neuron
			8/16	im x 2	Neuron
8/17	im x 2	Non-neuron
			8/18	im	Neuron

	Degree of depth spinal cord injection
			8/19	im	hES
8/23	im	Neuron hES
			8/24	im x 2	hES
8/25	im x 2	hES
			8/26	im x 2	Neuron
8/29	im x 2	Neuron
			8/31	im x 2	Neuron
9/5	im iv	Non-neuron
			9/7	im	Neuron
9/12	im	Neuron
			9/15	Epidural	Neuron
9/16	im	Neuron
			9/19	iv	Neuron
9/22	iv	Neuron
			9/28	im	Neuron
9/30	iv	Neuron
			10/3	im iv	Neuron
10/4	im iv	Non-neuron neuron and non-neuron mixture
			10/5	iv	Neuron
10/13	im	Neuron
			10/14	Intraarticular	Neuron
10/17	iv	Neuron
			11/15	iv	Neuron
11/18	im	Non-neuron
			11/21	iv	Non-neuron
11/23	im	Non-neuron
			12/1	im iv	Neuron and non-neuron mixture
12/6	im	Neuron and non-neuron mixture
			12/8	im	Non-neuron
12/13	im	The non-neuron neuron
			12/15	Epidural	Neuron
12/16	im	Neuron
			12/21	im	Neuron
12/22	im	Neuron

	iv
			12/25	im iv	Non-neuron
3/5	spinal	Non-neuron

Embodiment 11:

(John Hopkins University) is diagnosed as familial olivary body pons brain atrophy to this patient in the Johns Hopkins University.Her father, elder sister born of the same parents (younger sister) and brother also suffer from same disease.She can only talk when exhaling, can not leave wheelchair, urinates continuously and have to manual defecate.She trembles by whole body, even can not self balance when sitting.Serious postural hypotension is arranged.

Treat after 2 years, this patient can go to toilet alone, well talk, do not tremble and can walk having under the situation of support.Just do not worsen from treatment, although her elder sister born of the same parents (younger sister) is unable to leave the bed because of same disease.She does not have postural hypotension, basic not deterioration.

Treating for skin disease

Put into practice according to the present invention, by subcutaneous or intravenous injection or by local or local approach, give about 750,000-160,000,000 hES cell and/or its derivant are with the treatment dermatosis, include but not limited to ulcer, psoriasis, decubital ulcer and the sarcoidosis of disunion, wherein said cell comprises the hematopoietic stem cell CFU-GM.Under dermopathic situation, also can locally use the hES cell with treatment dermatosis or damage.In another embodiment, give about 750,000-80,000,000 cell.

In one embodiment, can be with Skin Cell and the embryo extract and the biological compatibility carrier of hES cell or differentiation, mix as gel, ointment or paste, and put on the wound that impaired skin or mucosal tissue are difficult to heal with healing acceleration and healing, as diabetic or decubital ulcer.Perhaps, this cell can suspension or emulsion form provide.In one embodiment, carrier also contains antimicrobial, analgesic or other medicament.

Perhaps, in a different embodiment, on artificial aseptic porous matrix such as muslin, cultivate the hES cell, be directly applied for wound.After 12-24 hour, take off this muslin, this wound is cured in the stem cell continued growth.When using in the damage, this hES cell can break up and the final damaged cell that replaces.

Embodiment 12:

Women left side ankle was burnt in 70 years old, can't cure with conventional therapy.With the hES cell be applied to the burn position after, her wound begin the healing, after 3 years, the skin complete recovery of health.

The treatment of autoimmune disease

Put into practice according to the present invention, give about 750 by intramuscular injection, intravenous injection, subcutaneous injection, intra-articular injection or intravenous infusion or its combination, 000-160,000,000 hES cell and/or its derivant, with the treatment autoimmune disease, include but not limited to: systemic lupus erythematosus (sle), ankylosing spondylitis, cardiomyopathy, sarcoidosis, arthritis and ulcerative colitis, wherein said cell comprises the hematopoietic stem cell CFU-GM.In another embodiment, give about 750,000-80,000,000 cell.Give the hES cell and can not only treat autoimmune disease highly effectively, and can the downtrod immune system of high efficient recovery.As the method that the patient of organ transplantation or the intervention of other medical science is carried out in alternative or " anticipating " preparation of immunosuppressant treatment, can utilize hES cell and its derivant to accept organ by reducing host-transplant rejection.

Though can change dosage regimen to be fit to concrete patient, but the typical scenario of treatment autoimmune disease is included in injection initial two months every days, injected every other day and intravenous infusion at three month, injected weekly at 5-7 month and 10-12 month and per 15 days continuous intravenous infusions 2 days, and in 1 year booster injection in per 3 months once, booster injection in per 6 months are once in 1 year then.

Embodiment 13:

Suffered from psoriasic patient in the past in 26 years and can not leave wheelchair, and suffer from hypertension, diabetes and psoriatic arthritis, disunion takes place on its left lower limb and be not obedient to dermatoplastic huge ulcer.

After receiving treatment 6 months, her ulcer and arthritis disappear.Her diabetes and hypertension are controlled.She begins autonomous walking, begins all activities, accepts the booster injection treatment in the past in 1 year.She claim oneself in the past 26 annual controls once walk, and can do all housework alone.

Wound is carried out local stem-cell therapy, and wound is cured.After the treatment, do not detect other wound.The scaliness of skin and gargalesthesia and lower extremity swelling and distension also alleviate.Also cured diabetes, blood pressure is controlled, causes medication to reduce.

This patient's infusion protocol is referring to table 12.

Table 12

Date	Route of administration	Cell type
			12/27	Proof load	Non-neuron
1/22	im	Non-neuron
			2/20	im	Neuron
2/23	im	Neuron
			2/28	m	Neuron
4/1	im	Neuron
			4/7	im	Neuron and non-neuron mixture
4/10	im	Neuron and non-neuron mixture
			4/21	m	Neuron and non-neuron mixture
6/29	im	Non-neuron
			7/6	m	Non-neuron
7/29	im	Non-neuron
			10/17	The im spraying	Non-neuron
11/1	The im spraying	Neuron and non-neuron mixture
			11/2	The im spraying	Neuron and non-neuron mixture
11/3	The im spraying	Neuron and non-neuron mixture
			11/4	im x 2	Neuron
11/5	The iv spraying	Non-neuron
			11/6	The im spraying	Non-neuron
11/7	The im spraying	Non-neuron
			11/11	The iv spraying	Neuron
11/14	The iv spraying	Neuron
			11/15	The im spraying	Non-neuron
11/16	im	Non-neuron
			11/21	iv	Non-neuron
11/23	im	Non-neuron
			11/29	im	Neuron and non-neuron mixture
1/4	im	Non-neuron
			3/28	im	Non-neuron

Hemopathic treatment

Another embodiment of the inventive method is by intravenous injection hES cell and/or its derivatives for treatment hematopathy, as thrombocytopenia, thalassemia, acute myeloid leukemia, wherein said cell comprises the hematopoietic stem cell CFU-GM.Give cell 10-14 days, then benign disease is overstated multiple injection weekly once.For malignant disorders, injection every day in 40 days is if recur then then duplicate injection.

The treatment of genetic diseases

According to the present invention's practice, the another kind of embodiment of hES cell transplantation method is the treatment genetic diseases, includes but not limited to: mongolism, Friedreich ataxia, Huntington chorea, Asperger syndrome and Duchenne-Arandisease.

The symptom of genetic diseases shows as mental retardation, flesh skeletal function obstacle and organ failure alone or in combination, causes the serious weak and reduction life expectancy that can not cure.In suffering from the patient of heredopathia, give this patient hES cell according to the present invention practice, after giving, this cell differentiation is also lost or the cell colony of damaged gene outcome for this patient provides to express, and recovers born of the same parents' homeostasis subsequently.

Though can change dosage regimen to be fit to concrete patient, but the typical scenario of treatment genetic diseases is included in intramuscular and intravenous injection every day first month, the injection every other day at 2nd month, injected weekly twice at three month, the 5th, 7,9,11 and 12 months weekly the injection once, the every three months booster injection are once in 1 year.In addition, carried out intravenous infusion in per 15 days.

Embodiment 14:

The mongolism patient is reactionless to verbal order, can not stair activity, because the difficult body weight of feed is very low.This patient can not take food, and demonstrates typical wide stance (wide stance) and coughs continually and catch a cold.Eyes are mongolism.

According to treatment of the present invention, this patient can understand also effective language order, loquitur and stair activity.It is littler 1 years old than her actual age that DQ test is presented in 1 year its mental age, and relative is 2 years old 6 months.Socially, she goes to school and plays with other child.She also can take food herself.

This patient's infusion protocol is referring to table 13.

Table 13

Date	Route of administration	Cell type
			7/8	Proof load	Non-neuron
7/16	im	Neuron
			7/25	im	Neuron
7/26	im	Neuron
			7/27	im	Neuron
7/30	im	Neuron
			8/3	im	Neuron
8/10	im	Neuron
			8/12	im	Neuron
8/16	im	Neuron
			8/18	im	Neuron
8/22	im	hES
			8/26	im	Neuron
8/29	im	Neuron
			8/31	im	Neuron
9/2	im	Neuron
			9/6	im	Neuron
9/8	im	Non-neuron
			9/13	im	Non-neuron
9/19	im	Neuron and non-neuron mixture
			9/22	im	Non-neuron
9/30	im	Non-neuron
			10/3	im	Neuron and non-neuron mixture
10/10	im	Non-neuron
			10/18	im	Non-neuron
10/27	im	Neuron
			11/7	im	Non-neuron
11/8	im	Non-neuron
			11/9	im	Non-neuron
11/10	im	Non-neuron
			11/11	im	Neuron
11/14	im	Neuron
			11/17	im	Non-neuron
11/18	im	Non-neuron
			11/21	im	Non-neuron
11/22	im	Non-neuron
			11/23	im	Non-neuron

11/25	im	Non-neuron
			11/28	im	Neuron and non-neuron mixture
12/4	im	Neuron and non-neuron mixture
			12/8	im	Non-neuron
12/12	im	Non-neuron
			12/15	im	Neuron
12/19	im	Neuron
			12/27	iv	Neuron
1/4	im iv	Non-neuron
			1/11	im	The non-neuron neuron
1/19	im	Neuron
			1/20	im	Non-neuron
1/23	im	Non-neuron
			1/30	im	Neuron
2/9	im	Neuron
			2/13	im	Neuron
2/23	im	Neuron
			2/27	im	Neuron
3/10	im	Neuron
			3/14	im	Neuron
3/20	im	Neuron
			5/11	im	Neuron
5/15	im	Neuron
			5/16	im	Neuron
5/17	im	Neuron
			5/18	im	Neuron
5/22	im	Neuron
			5/23	im	Neuron
7/3	im	Neuron
			7/5	im	Neuron
7/11	im	Neuron
			7/13	im	Neuron
7/17	im	Neuron
			7/19	im	Neuron
7/21	iv	Neuron
			7/24	im	Neuron
7/28	im	Non-neuron

7/31	im	Neuron
			8/2	im	Non-neuron
8/21	im	Non-neuron
			8/23	im	Non-neuron
8/28	im	Non-neuron
			8/30	im	Non-neuron

Embodiment 15:

3 years old girl is suffered from mongolism by diagnosis.She can not talk, understanding or stair activity.She can't take food alone, and the limited pronunciation of her word is very unclear.

Treat after 8 months, she can understand all things, says the sentence of three speech, and energy identification colors, shape and size.She is stair activity alone, and beginning feed alone.

This patient's infusion protocol is referring to table 14.

Table 14

Date	Route of administration	Cell type
			1/30	im	Neuron (proof load)
2/6	im	Neuron
			2/7	im	Neuron
2/8	im	Neuron
			2/9	im	Neuron
2/10	im	Non-neuron
			2/13	im	Neuron
2/15	im	Neuron
			2/16	im	Neuron
2/17	im	Neuron
			2/20	im	Neuron
2/22	im	Neuron
			2/24	im	Non-neuron
2/27	im	Neuron
			3/1	im	Neuron
3/3	im	Non-neuron
			3/6	im	Non-neuron
3/13	im	Neuron
			3/15	im	Neuron
3/22	im	Neuron
			3/24	im	Neuron
3/26	im	Neuron

3/27	im	Non-neuron
			3/29	im	Neuron
4/3	im	Neuron
			4/5	im	Neuron
4/7	im	Neuron
			4/10	im	Neuron
4/12	im	Non-neuron
			4/14	im	Non-neuron
4/21	im	Neuron
			4/24	im	Neuron
4/26	im	Neuron
			4/28	im	Neuron
5/1	im	Neuron
			5/10	im	Neuron
5/12	im	Neuron
			5/13	im	Non-neuron
5/22	im	Neuron
			5/24	im	Neuron
5/29	im	Neuron
			6/5	im	Neuron
6/7	im	Neuron
			6/10	im	Neuron
6/14	im	Neuron
			6/16	im	Neuron
6/19	im	Neuron
			6/21	im	Neuron
6/23	iv	Non-neuron
			6/27	im	Neuron
6/29	im	Neuron
			7/3	im	Neuron
7/5	im	Neuron
			7/12	im	Neuron
7/14	im	Neuron
			7/17	im	Neuron
7/19	im	Neuron
			7/21	im	Neuron
7/27	im	Neuron
			7/28	im	Non-neuron
8/1	im	Non-neuron

8/3	im	Non-neuron
			8/5	im	Non-neuron
8/7	im	Non-neuron
			8/19	im	Non-neuron
8/24	im	Non-neuron
			8/26	im	Neuron
8/28	im	Non-neuron
			8/31	im	Non-neuron
9/2	im	Non-neuron
			9/7	im	Non-neuron
9/9	im	Non-neuron
			9/11	im	Non-neuron
9/14	im	Non-neuron
			9/21	im	Non-neuron
9/23	im	Non-neuron
			9/25	im	Non-neuron
9/28	im	Non-neuron
			9/29	im	Non-neuron
10/4	im	Non-neuron
			10/7	im	Non-neuron
10/10	im	Non-neuron
			10/14	im	Non-neuron
10/16	im	Non-neuron
			10/18	im	Non-neuron
10/22	im	Non-neuron
			10/26	im	Non-neuron
10/28	im	Non-neuron
			11/1	im	Non-neuron
11/4	im	Non-neuron
			11/7	im	Non-neuron
11/9	im	Non-neuron
			11/13	im	Non-neuron
11/16	im	Non-neuron
			11/17	im	Non-neuron
11/20	im	Non-neuron
			11/23	im	Non-neuron
11/27	im	Non-neuron
			11/30	im	Non-neuron
12/3	im	Non-neuron

12/4	im x 2	Non-neuron
			12/7	im	Non-neuron
12/9	im	Non-neuron
			12/11	im	Non-neuron
12/14	im x 2	Non-neuron
			12/16	im	Non-neuron
12/21	im	Non-neuron
			12/24	im	Non-neuron
12/27	im	Neuron
			1/1	im	Mix
1/3	im	Non-neuron
			1/6	im	Non-neuron
1/9	im	Non-neuron
			1/12	im	Non-neuron
1/15	im	Non-neuron
			1/18	im	Non-neuron
1/22	im	Mix
			1/25	im	Neuron
1/28	im	Neuron

Embodiment 16:

13 years old boy suffers from genetic defect, attention-deficient, frequent spasm and visual impairment.He has complete tubular visual field.Even exclusive also being difficult to takes a seat on the classroom and looks at the blackboard before being sitting in.His critical event (milestone) postpones, if he reads, he has to read and just can remember the content of being read for three times.He also is difficult to the article in the orientation room.

Behind beginning hES cell therapy, his vision improves, and he is sitting in back two rows in the classroom and can more clearly sees blackboard clearly.He can remember more thing, and he improved to the corresponding time of verbal order.His spasm is not so much in the past, and its medication also reduces to some extent.The ophthalmologist reports that the confined area (deadened area) of its visual field (periphery) recovers.

This patient's infusion protocol is referring to table 15.

Table 15

Date	Route of administration	Cell type
			6/8	im	Neuron (proof load)
6/9	im	Neuron
			6/12	im	Neuron

6/13	im	Neuron
			6/14	im	Non-neuron
6/15	im	Non-neuron
			6/16	im	Neuron
6/20	im	Neuron
			6/21	im	Non-neuron
6/22	im	Neuron
			6/23	iv	Non-neuron
6/27	im	Neuron
			6/28	im	Neuron
6/29	im	Neuron
			7/3	im	Non-neuron
7/4	im	Neuron
			7/5	im	Neuron
7/6	im	Neuron
			7/7	im	Neuron
7/10	im	Neuron
			7/11	im	Neuron
7/12	im	Neuron
			7/13	im	Non-neuron
7/14	im	Neuron
			7/17	im	Neuron
7/18	im	Neuron
			7/20	im	Neuron
7/21	The iv infusion	Neuron
			7/22	The iv infusion	Neuron
7/24	im	Neuron
			7/25	im	Neuron
7/26	im	Neuron
			7/27	im	Neuron
7/28	im	Non-neuron
			7/31	im	Non-neuron
8/2	im	Non-neuron
			8/3	im	Non-neuron
8/4	im	Non-neuron
			8/7	im	Non-neuron
8/8	im	Non-neuron
			8/9	im	Non-neuron
8/10	im x 2	Non-neuron

8/11	The iv infusion	Non-neuron
			8/12	The iv infusion	Non-neuron
8/13	im	Non-neuron
			8/14	im	Non-neuron
8/17	im x 2	Non-neuron
			8/18	im	Non-neuron
8/21	im	Non-neuron
			8/22	im	Non-neuron
8/23	im	Non-neuron
			8/24	im	Non-neuron
8/25	im	Non-neuron
			8/2/	im	Non-neuron
8/29	im	Non-neuron
			8/30	im	Non-neuron
9/2	The iv infusion	Non-neuron
			9/3	The iv infusion	Non-neuron
9/4	im x 3	Non-neuron
			9/5	im	Non-neuron
9/6	im	Non-neuron
			9/7	im	Non-neuron
9/8	im	Non-neuron
			9/11	im	Non-neuron
9/13	im	Non-neuron
			9/14	im	Non-neuron
9/16	im	Non-neuron
			9/18	im	Non-neuron
9/19	im	Non-neuron
			9/21	im x 2	Non-neuron
9/22	The iv infusion	Non-neuron
			9/23	The iv infusion	Non-neuron
10/3	im	Non-neuron
			10/4	im	Non-neuron
10/5	im	Non-neuron
			10/6	im	Non-neuron
10/9	im	Non-neuron
			10/10	im	Non-neuron
10/11	im	Non-neuron
			10/12	im	Non-neuron
10/13	Behind the eye socket	Neuron

10/16	im	Non-neuron
			10/17	im	Non-neuron
10/18	im	Non-neuron
			10/20	im	Non-neuron
10/23	im	Non-neuron
			10/24	im	Non-neuron
10/25	im	Non-neuron
			10/26	im	Non-neuron
10/27	The iv infusion	Non-neuron
			10/28	Iv infusion x3	Non-neuron
10/31	im x 2	Non-neuron
			11/2	im x 2	Non-neuron
11/3	im x 2	Non-neuron
			11/6	im x 2	Non-neuron
11/8	im	Non-neuron
			11/9	im	Non-neuron
11/10	im	Non-neuron
			11/13	im	Non-neuron
11/14	Behind the eye socket	Neuron
			11/15	im	Non-neuron
11/16	im	Non-neuron
			11/17	im	Non-neuron
11/20	im	Non-neuron
			11/21	im	Non-neuron
11/23	im	Non-neuron
			11/24	im	Non-neuron
11/29	im	Non-neuron
			11/30	iv	Mix
12/1	The iv infusion	Mix
			12/4	im	Non-neuron
12/5	iv	Mix
			12/6	im	Non-neuron
12/7	im	Non-neuron
			12/8	im	Non-neuron
12/11	im	Non-neuron
			12/12	im	Non-neuron
12/13	im	Non-neuron
			12/14	im	Non-neuron
12/18	im	Non-neuron

12/19	iv	Non-neuron
			12/21	im	Non-neuron
12/22	Behind the eye socket	Neuron
			12/29	im	Neuron
2/2	im	Non-neuron

Embodiment 17:

This patient is the 32 years old male who suffers from Friedreich ataxia, and its elder sister (younger sister) dies from this disease.He can not stand, speech is weak, be difficult to swallow, can not leave wheelchair and go to toilet and want help.Receive treatment in the Germany and the U.S. before him, but come to nothing.His cardiac dilatation, functional level are 15-20%.

Behind beginning hES cell therapy, he has occurred improving.He can more clearly talk, and does not have dysphagia.His heart has improvement, and all sizes are got back to normal value, and the function capacity is increased to 58-60%.Do not worsen.

This patient's infusion protocol is referring to table 16.

Table 16

Date	Route of administration	Cell type
			2/17	im	Non-neuron (proof load)
2/20	im iv	The neuron non-neuron
			2/21	im iv	The neuron non-neuron
2/22	im iv	The neuron non-neuron
			2/23	im iv	The neuron non-neuron
2/24	im iv	The neuron non-neuron
			2/25	im iv	The neuron non-neuron
2/26	im iv	The neuron non-neuron
			2/27	Epidural	Neuron
2/28	im iv	The neuron non-neuron
			3/1	im	Neuron

	iv	Non-neuron
			3/2	im iv	The neuron non-neuron
3/3	im iv	The neuron non-neuron
			3/4	im iv	The neuron non-neuron
3/5	im iv	The neuron non-neuron
			3/6	im	The neuron non-neuron
3/7	Epidural (conduit)	Neuron
			3/8	Epidural (conduit)	Neuron
3/9	Epidural (conduit)	Neuron
			3/10	Epidural (conduit)	Neuron
3/13	im iv	The neuron non-neuron
			3/14	im	Non-neuron
3/16	im	Non-neuron
			3/17	im iv	The neuron non-neuron
3/18	im iv	The neuron non-neuron
			3/19	im iv	The neuron non-neuron
3/20	im iv	The neuron non-neuron
			3/21	im	Non-neuron
3/22	im	Non-neuron
			3/24	im iv	The neuron non-neuron
3/25	im	Non-neuron
			3/26	im	Non-neuron
3/27	im	Neuron
			3/28	im	Non-neuron
3/29	im	Non-neuron
			3/30	im	Non-neuron
3/31	im	Non-neuron
			4/1	im	Non-neuron

4/2	im	Non-neuron
			4/3	im	Non-neuron
4/4	im iv	The neuron non-neuron
			4/5	im	Non-neuron
4/6	im	Non-neuron
			4/7	im	Non-neuron
4/8	im	Non-neuron
			4/9	im iv	The neuron non-neuron
4/10	im iv	The neuron non-neuron
			4/11	im	Non-neuron
4/15	im	Non-neuron
			4/16	im	Non-neuron
4/18	im iv	The neuron non-neuron
			4/19	im iv	The neuron non-neuron
4/20	im iv	The neuron non-neuron
			4/21	im	Non-neuron
4/22	im	Non-neuron
			4/24	im	Non-neuron
4/25	im	Non-neuron
			4/26	iv im	The neuron non-neuron
4/27	iv im	The neuron non-neuron
			4/29	iv	Neuron
4/30	iv im	The neuron non-neuron
			502	im	Non-neuron
5/3	iv	Neuron
			5/5	The iv epidural	Neuron
5/7	iv im	The neuron non-neuron
			5/9	iv im	The neuron non-neuron

5/10	iv im	The neuron non-neuron
			5/12	iv im	The neuron non-neuron
5/13	iv im	The neuron non-neuron
			5/14	iv im	The neuron non-neuron
5/16	Epidural (in the sheath)	Neuron
			5/18	iv im	The neuron non-neuron
8/26	im iv	Non-neuron
			8/27	im iv	Non-neuron
8/28	Iv iv infusion	Non-neuron
			8/29	Iv iv infusion	Non-neuron
8/30	im iv	Non-neuron
			8/31	im iv	Non-neuron
9/4	im iv	Non-neuron
			9/6	im iv	Non-neuron
9/7	im iv	Non-neuron
			9/9	im iv	Non-neuron
9/10	Epidural (in the sheath)	Neuron
			9/12	im iv	Non-neuron
9/14	im iv	Non-neuron
			9/15	Epidural (conduit)	Neuron,
9/16	Epidural (conduit)	Neuron,
			9/17	Epidural (conduit)	Neuron
9/18	im iv	Non-neuron
			9/19	im iv	Non-neuron

9/20	Iv iv infusion	Non-neuron
			9/21	Iv iv infusion	Non-neuron

The treatment of oculopathy

Put into practice according to the present invention, the Another Application of hES cell transplantation is according to the present invention practice neuron progenitor cell and hES cell to be injected directly into the eyeball rear of eyes, the shallow table section of eyes and the inner chamber of eyes to treat oculopathy, includes but not limited to: optic atrophy, degeneration of macula, ocular injury, corneal graft rejection and retinitis pigmentosa.

Though can change dosage regimen to be fit to concrete patient, but the typical scenario of treatment oculopathy comprised intramuscular and intravenous injection 10 days, it is behind the eyeball then or intravitreal injection, intramuscular and intravenous injection 15 days, be retrobulbar injection then, 2 intravenous infusions carry out retrobulbar injection after 2 months.Amount to retrobulbar injection 4 times in the time in 8 months-1 year, observed result then is in some cases in early stage observed result.Can utilize approach in the vitreous body.In addition, can there be the contact lens of stem cell to come repairing corneal by using growth.Also can use the eye drop that contains stem cell.

Embodiment 18:

Diagnosis has the patient of optic atrophy to show following disease, comprises reading the small character difficulty, can't see distant objects clearly.Can not see navy blue and purple.Vision is zero, and the energy sensing light is 6/24 now.

According to stem cell therapy of the present invention, promptly, hES cell and its derivant of comprising the neuronal stem cell CFU-GM by retrobulbar injection, put into practice according to the present invention, give by local intravenous injection, subcutaneous injection or intramuscular injection, intravitreal injection or local application about 750,000-160,000,000 hES cell and its derivant that comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM, this patient demonstrates remarkable improvement.He can readout time, can see billboard, and can see TV outside certain distance.

This patient's infusion protocol is referring to table 17.

Table 17

Date	Route of administration	Cell type
			2/14	im	Neuron
2/15	iv	The neuron non-neuron
			3/20	iv x 2 im	Neuron
3/21	Behind the iv eyeball	Neuron
			4/30	iv	Non-neuron
5/1	iv im	Neuron
			5/2	Behind the eyeball	Non-neuron

The treatment of hepatopathy and nephropathy

According to the present invention practice, it is to put into practice to transplant according to the present invention to treat hepatopathy and nephropathy that the another kind of hES cell transplantation is used, and includes but not limited to: latter stage nephroptosis and nephrotic syndrome.Route of administration is intravenous, intramuscular and intravenous infusion.

Embodiment 19:

Diagnosis has the patient of nephrotic syndrome to show carbamide, inosine and the rising of P protein level.Can not keep the differentiation of skin medullary substance, health swelling.Put into practice according to the present invention, give by intravenous injection, subcutaneous injection, intramuscular injection or intravenous infusion about 750,000-160,000,000 hES cell and its derivant comprise the hematopoietic stem cell CFU-GM, produce albumin stem cell CFU-GM and produce bilirubin stem cell CFU-GM.Behind stem-cell therapy, the swelling degree of health alleviates, and the differentiation of skin medullary substance is recovered.Carbamide, inosine and P protein level also recover standard.

This patient's infusion protocol is referring to table 18.

Table 18

Date	Route of administration	Cell type
			8/10	Proof load	Neuron
8/11	im	Neuron
			8/12	im	Neuron
8/16	im	hES
			8/22	im	hES
8/24	im	hES

8/25	im	Neuron
			8/29	im	Neuron
9/1	im	Neuron
			9/7	im	Neuron
9/8	im	Non-neuron
			9/12	im	Neuron and non-neuron mixture
9/19	im	Neuron and non-neuron mixture
			10/3	im	Neuron and non-neuron mixture
10/10	im	Neuron and non-neuron mixture
			10/11	iv	Neuron and non-neuron mixture
10/31	iv	Non-neuron
			11/10	im	Non-neuron
11/21	im	Non-neuron
			12/8	im	Non-neuron
12/23	iv	Non-neuron
			2/7	im	Non-neuron
8/24	im	Non-neuron
			8/25	im	Non-neuron
9/9	im	Non-neuron

The treatment of flesh-skeletal diseases

According to the present invention's practice, it is treatment flesh-skeletal diseases that the another kind of hES cell transplantation is used, and includes but not limited to: arthritis, Duchenne's dystrophy, limb-girdle muscular dystrophy, spinal cord muscular dystrophy and becker's dystrophy.

Though can change dosage regimen to adapt to concrete patient, but the typical scenario of treatment flesh-skeletal diseases is included in intramuscular or intravenous injection every day first month, the 3rd, 5,6 with injected twice weekly in 7 months, injection weekly in 9-12 month, booster injection in per 3 months once.Give one time intravenous infusion in every 10-15 days, gave the intramuscular injection of a degree of depth spinal cord in every 15-30 days.

Embodiment 20:

Diagnosis has patient's the medical history of Duchenne's dystrophy to show following symptom, for example tired, thigh and facial swelling, and positive Gao Er sign, the high-level inosinic acid kinases (CPK) of 10,000 units.

By intravenous injection, subcutaneous injection, intramuscular injection, intravenous catheter infusion, epidural catheter infusion or subarachnoid block conduit infusion, with hES cell and this patient of its derivatives for treatment after the scheduled time, its CPK level significantly is reduced to 1198 units, the Gao Er sign is negative, and described hES cell and its derivant comprise neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM alone or in combination.

This patient's infusion protocol is referring to table 19.

Table 19

Date	Route of administration	Cell type
			3/18	Proof load	Non-neuron
3/19	im	Neuron
			3/20	im x 2	Neuron
3/25	im x 2	Non-neuron
			3/26	im x 2	Non-neuron
3/27	im x 4	Neuron x 2 non-neuron x 2
			3/28	im x 4	Neuron x 2 non-neuron x 2
3/29	im x 4	Neuron x 2 non-neuron x 2
			3/30	im x 4	Neuron x 2 non-neuron x 2
3/31	im x 4	Neuron non-neuron x 3
			4/1	im x 4	Neuron x 2 non-neuron x 2
4/2	im x 3	Neuron non-neuron x 2
			4/3	im x 4	Neuron x 2 non-neuron x 2
4/4	im x 4	Neuron x 2 non-neuron x 2
			4/5	im x 4	Neuron x 2 non-neuron x 2
4/6	im x 4	Neuron x 2 non-neuron x 2
			4/7	im x 4	Neuron x 2 non-neuron x 2
4/8	im x 4	Neuron x 2 non-neuron x 2
			4/9	im x 4	Neuron x 2

		Non-neuron x 2
			4/10	im x 4	Neuron x 2 non-neuron x 2
4/11	im x 4	Neuron x 2 non-neuron x 2
			4/12	im x 4	Neuron x 2 non-neuron x 2
4/13	im x 4	Neuron x 2 non-neuron x 2
			4/14	im x 4	Neuron x 2 non-neuron x 2
4/15	im x 4	Neuron x 2 non-neuron x 2
			4/16	im x 4	Neuron x 2 non-neuron x 2
4/17	im x 5	Neuron x 3 non-neuron x 2
			4/18	im x 3	Neuron non-neuron x 2
4/19	im x 3	Neuron x 2 non-neurons
			4/20	im x 3	Neuron non-neuron x 2
4/21	im x 3	Neuron non-neuron x 2
			4/22	im x 3	Neuron non-neuron x 2
4/23	im x 3	Neuron x 2 non-neurons
			4/24	im x 4	Neuron x 2 non-neuron x 2
4/25	im x 3	Neuron non-neuron x 2
			4/26	im x 2	The neuron non-neuron
7/1	im x 2	The neuron non-neuron
			7/2	im x 4	Neuron x 2 non-neuron x 2
7/3	im x 4	Neuron x 2

		Non-neuron x 2
			7/4	im x 2	The neuron non-neuron
7/5	Iv infusion x 2	Neuron
			7/6	Iv infusion x 2	Neuron
7/7	im x 2	Neuron
			7/8	im x 2	Neuron
7/9	im x 2	The neuron non-neuron
			7/10	im x 2	The neuron non-neuron
7/11	im x 2	The neuron non-neuron
			7/12	im x 2	The neuron non-neuron
7/13	im x 2	The neuron non-neuron
			7/14	im x 2	The neuron non-neuron
7/15	im x 2	The neuron non-neuron
			7/16	im x 3	Neuron
7/17	Iv infusion im x 2	The neuron non-neuron
			7/18	Iv infusion im x 2	The neuron non-neuron
7/19	im x 4	Neuron x 2 non-neuron x 2
			7/20	im x 4	Neuron x 2 non-neuron x 2
7/21	im x 4	Neuron x 2 non-neuron x 2
			7/22	im x 4	Neuron x 2 non-neuron x 2
7/23	iv x 2 im x 4	Neuron x 2 non-neuron x 4
			7/24	im x 4	Neuron x 2 non-neuron x 2
7/25	iv im x 4	Neuron x 2 non-neuron x 4

7/26	im x 4	Neuron x 2 non-neuron x 2
			7/27	iv im x 4	Neuron x 2 non-neuron x 4
7/28	im x 4	Neuron non-neuron x 3
			7/29	iv im x 4	Non-neuron
7/30	im x 4	Neuron x 2 non-neuron x 2
			7/31	iv im x 4	Neuron x 2 non-neuron x 3
8/1	im x 4	Neuron x 2 non-neuron x 2
			8/2	im x 4	Non-neuron
8/3	Iv infusion x 2	Non-neuron
			8/4	Iv infusion x 2	Non-neuron
8/5	im x 4	Non-neuron
			8/6	im x 4	Non-neuron
8/7	Iv infusion im x 2	Non-neuron
			8/8	Iv infusion im x 2	Non-neuron
8/9	Iv infusion x 2 im x 2	Non-neuron
			8/10	im x 4	Non-neuron
8/11	im x 4	Non-neuron
			8/12	Iv infusion im x 2	Neuron

Embodiment 21:

Diagnosis has 14 years old boy of DMD to be unable to leave the bed, the hands skelasthenia, and muscular dystrophy is simultaneously with skoliosis.

Treat after 8 months, patient's biceps, triceps muscle and brachioradialis grow to some extent, and arm can be lifted high to the ancon level.Body weight gain, and grow tall, and without any relevant skoliosis.His CPK level (showing the muscle degradation rate) also reduces.

This patient's infusion protocol is referring to table 20.

Table 20

Date	Route of administration	Cell type
			4/13	im	Non-neuron (proof load)
4/14	im	Non-neuron
			4/15	im	Neuron
4/16	im	Neuron
			4/17	im	The neuron non-neuron
4/18	im	Non-neuron
			4/19	im	The neuron non-neuron
4/20	im	The neuron non-neuron
			4/21	im	The neuron non-neuron
4/22	im	The neuron non-neuron
			4/23	im	The neuron non-neuron
4/24	im	The neuron non-neuron
			4/25	im	The neuron non-neuron
4/26	im	Non-neuron
			4/27	im	Non-neuron
4/28	im	The neuron non-neuron
			4/29	im	The neuron non-neuron
4/30	im	The neuron non-neuron
			5/1	im	Non-neuron
5/2	im	Non-neuron
			5/3	im	The neuron non-neuron
5/4	im	Non-neuron
			5/5	im	The neuron non-neuron
5/6	im	Neuron

5/7	im	The neuron non-neuron
			5/8	im	The neuron non-neuron
5/9	Iv infusion im	Neuron
			5/13	im	The neuron non-neuron
5/15	im	Non-neuron
			5/16	im	Non-neuron
5/17	im	The neuron non-neuron
			5/18	im	The neuron non-neuron
5/19	im	The neuron non-neuron
			5/20	im	The neuron non-neuron
5/21	im	The neuron non-neuron
			5/22	im	The neuron non-neuron
5/23	im	The neuron non-neuron
			5/24	im	The neuron non-neuron
5/26	im	The neuron non-neuron
			5/27	im	The neuron non-neuron
5/28	im	The neuron non-neuron
			5/29	im	The neuron non-neuron
5/30	im	The neuron non-neuron
			6/1	im	The neuron non-neuron
6/2	im	The neuron non-neuron

6/3	im	The neuron non-neuron
			6/4	im	The neuron non-neuron
6/5	im	The neuron non-neuron
			6/6	im	The neuron non-neuron
6/7	im	The neuron non-neuron
			6/8	im	The neuron non-neuron
6/9	im	The neuron non-neuron
			6/10	im	The neuron non-neuron
6/11	im	The neuron non-neuron
			6/12	im	Non-neuron
6/13	im	The neuron non-neuron
			6/14	im	The neuron non-neuron
6/15	im	The neuron non-neuron
			6/16	im	The neuron non-neuron
6/17	im	The neuron non-neuron
			6/18	im	The neuron non-neuron
6/19	im	Neuron x 2
			6/20	im	The neuron non-neuron
6/22	im	Neuron x 2
			6/23	iv	Non-neuron x 2
6/24	iv	Non-neuron x 2
			6/25	iv	Non-neuron x 2
6/27	im	The neuron non-neuron

6/28	im	The neuron non-neuron
			6/29	im	The neuron non-neuron
6/30	im	The neuron non-neuron
			7/1	im	The neuron non-neuron
7/2	im	The neuron non-neuron
			7/3	im	The neuron non-neuron
7/4	im	The neuron non-neuron
			7/5	im	Neuron x 2
7/6	im	Neuron x 2
			7/7	im	Neuron x 2
7/8	im	The neuron non-neuron
			7/9	im	The neuron non-neuron
7/10	im	The neuron non-neuron
			7/11	im	The neuron non-neuron
7/12	im	The neuron non-neuron
			7/13	im	The neuron non-neuron
7/14	im	The neuron non-neuron
			7/15	im	The neuron non-neuron
8/18	im	Non-neuron x 3
			8/19	im	Non-neuron x 2
8/20	im	Non-neuron x 3
			8/21	im	Non-neuron x 3
8/22	im	Non-neuron x 3
			8/23	im	Non-neuron x 3
8/24	im	Non-neuron x 3

8/25	im	Neuron non-neuron x 2
			8/26	im	Neuron x 3
8/27	im	Non-neuron x 3
			8/28	iv	Non-neuron 7
8/29	iv	Non-neuron 7x 2
			8/30	im	Non-neuron x 2
8/31	im	Non-neuron x 3
			9/1	The iv infusion	Non-neuron
9/2	The iv infusion	Non-neuron
			9/3	im	Non-neuron x 3
9/4	im	Non-neuron x 3
			9/5	im	Non-neuron x 3
9/6	im	Non-neuron x 3
			9/7	im	Non-neuron x 3
9/8	im	Non-neuron x 3
			9/9	im	Non-neuron x 3
9/10	im	Non-neuron x 3
			9/11	im	Non-neuron x 3
9/12	im	Non-neuron x 3
			9/13	im	Non-neuron x 3
9/14	im	Non-neuron x 3
			9/15	im	Non-neuron x 3
9/16	im	Non-neuron x 3
			9/17	im	Non-neuron x 3
9/18	im	Non-neuron x 3
			9/19	im	Non-neuron x 3
9/20	im	Non-neuron x 3
			9/21	im	Non-neuron x 3
9/22	The iv infusion	Non-neuron
			9/23	The iv infusion	Non-neuron
9/24	im	Non-neuron x 3
			9/25	im	Non-neuron x 3
9/26	im	Non-neuron x 3
			9/27	im	Non-neuron x 3
9/28	im	Non-neuron x 3
			9/29	im	Non-neuron x 3
9/30	im	Non-neuron x 3
			10/1	im	Non-neuron x 3

10/2	im	Non-neuron x 3
			10/3	im	Non-neuron x 3
10/4	im	Non-neuron x 3
			10/5	im	Non-neuron x 3
10/6	im	Non-neuron x 3
			10/7	im	Non-neuron x 3
10/8	im	Non-neuron x 3
			10/9	im	Non-neuron x 3
10/11	im	Non-neuron x 3
			10/12	im	Non-neuron x 3
10/13	im	Non-neuron x 3
			10/14	im	Non-neuron x 3
10/15	im	Non-neuron x 3
			10/16	im	Non-neuron x 3
10/17	im	Non-neuron x 3
			10/18	im	Non-neuron x 3
10/19	im	Non-neuron x 3
			10/20	im	Non-neuron x 3
10/21	im	Non-neuron x 3
			10/23	im	Non-neuron x 3
10/24	im	Non-neuron x 3
			10/25	im	Non-neuron x 3
10/26	im	Non-neuron x 3
			10/27	im	Non-neuron x 3
10/28	im	Non-neuron x 3
			10/29	im	Non-neuron x 3
10/30	im	Non-neuron x 3
			10/31	im	Non-neuron x 5
11/2	im	Non-neuron x 4
			11/3	im	Non-neuron x 3
11/4	im	Non-neuron x 3
			11/5	im	Non-neuron x 3
11/7	im	Non-neuron x 3
			11/8	im	Non-neuron x 2
11/9	im	Non-neuron
			11/10	im	Non-neuron x 3
11/11	im	Non-neuron x 3
			11/12	im	Non-neuron x 3
11/13	im	Non-neuron x 3

11/14	im	Non-neuron x 3
			11/15	im	Non-neuron x 3
11/30	im	Mix x 3
			12/1	im	Non-neuron x 3
12/2	im	Non-neuron x 3
			12/3	im	Non-neuron x 3
12/4	im	Non-neuron x 3
			12/5	im	Non-neuron x 3
12/6	im	Non-neuron x 3
			12/7	im	Non-neuron x 3
12/8	im	Non-neuron x 3
			12/9	im	Non-neuron x 3
12/10	im	Non-neuron x 3
			12/12	im	Non-neuron x 3
12/13	im	Non-neuron x 3
			12/14	im	Non-neuron x 3
12/15	im	Non-neuron x 3
			12/16	The iv infusion	Non-neuron
12/17	Iv infusion im	Non-neuron x 3
			12/18	im	Non-neuron
12/19	iv im	Non-neuron
			12/20	im	Non-neuron x 3
12/21	iv im	Non-neuron x 3
			12/22	iv	Non-neuron x 3
12/23	im	Non-neuron x 3
			12/24	im	Non-neuron x 3
12/25	im	Mix x 3
			12/26	iv im	Non-neuron x 3
12/27	iv	Mix
			12/28	im	Non-neuron x 3
12/29	im	Non-neuron x 3
			12/30	im	Non-neuron
12/31	im	Non-neuron x 3
			1/1	im	Mix x 3
1/2	im	Mix x 3
			1/3	im	Non-neuron x 3

1/4	im	Non-neuron x 3
			1/5	The iv infusion	Non-neuron
1/6	The iv infusion	Non-neuron
			1/7	im	Non-neuron x 3
1/8	im	Non-neuron x 3
			1/9	im	Non-neuron x 3
1/10	im	Non-neuron x 3
			1/11	im	Non-neuron x 3
1/12	im	Non-neuron x 3
			1/13	im	Non-neuron x 3
1/14	im	Non-neuron x 3
			1/15	im	Non-neuron x 3
1/16	The iv infusion	Mix
			1/17	The iv infusion	Mix
1/18	im	Non-neuron
			1/19	im	Non-neuron
1/20	im	Non-neuron
			1/21	The iv infusion	Mix
1/22	im	Mix
			1/23	im	Mix
1/24	im	Neuron
			1/25	im	Neuron
1/26	im	Mix
			1/27	im	Mix
1/28	im	Neuron x 3
			1/29	The iv infusion	Mix
1/30	The iv infusion	Non-neuron

Embodiment 22:

Because DMD can not leave 8 years old boy of wheelchair and can't walk or stand.Treat after 6 months, his CPK level reduces.He does not worsen, and can move its arm.He begins to walk under by situation about seldom supporting.

This patient's infusion protocol is referring to table 21.

Table 21

Date	Route of administration	Cell type
			4/29	im	The neuron non-neuron
4/30	im	Non-neuron
			5/1	im	The neuron non-neuron
5/3	im	Neuron
			5/4	im	The neuron non-neuron
5/5	im	The neuron non-neuron
			5/6	im	The neuron non-neuron
5/7	im	Non-neuron
			5/8	im	The neuron non-neuron
5/9	im	The neuron non-neuron
			5/10	im	The neuron non-neuron
5/11	im	The neuron non-neuron
			5/12	im	The neuron non-neuron
5/13	im	The neuron non-neuron
			5/14	im	The neuron non-neuron
5/15	im	The neuron non-neuron
			5/16	im	The neuron non-neuron
5/17	im	The neuron non-neuron
			5/18	im	The neuron non-neuron
5/19	im	The neuron non-neuron

5/20	im	The neuron non-neuron
			5/21	im	The neuron non-neuron
5/22	im	The neuron non-neuron
			5/23	im	The neuron non-neuron
5/24	im	The neuron non-neuron
			5/25	im	The neuron non-neuron
5/26	im	The neuron non-neuron
			5/27	im	The neuron non-neuron
5/28	im	The neuron non-neuron
			5/29	im	The neuron non-neuron
5/30	im	The neuron non-neuron
			6/1	im	The neuron non-neuron
6/2	im	The neuron non-neuron
			6/3	im	The neuron non-neuron
6/4	im	The neuron non-neuron
			6/5	im	The neuron non-neuron
6/6	im	The neuron non-neuron
			6/7	im	The neuron non-neuron
6/8	im	The neuron non-neuron
			6/9	im	The neuron non-neuron

11/15	im	Non-neuron
			11/16	Intravenous infusion	Neuron
11/17	Intravenous infusion	Neuron
			11/18	im	Non-neuron
11/19	im	Non-neuron
			11/20	im	Non-neuron
11/21	Intravenous infusion	Mix
			11/22	Intravenous infusion	Mix
11/23	im	Non-neuron
			11/25	im	Mix
11/26	im	Mix
			11/27	im	Mix
11/29	im	Non-neuron
			12/1	im	Non-neuron
12/2	im	Non-neuron
			12/3	im	Non-neuron
12/5	im	Non-neuron
			12/6	im	Non-neuron
12/7	im	Non-neuron
			12/8	Intravenous infusion	Mix
12/9	Intravenous infusion	Non-neuron
			12/10	im	Non-neuron
12/11	im	Non-neuron
			12/12	im	Non-neuron
12/15	im	Non-neuron
			12/16	Intravenous infusion	Non-neuron
12/17	Intravenous infusion	Non-neuron
			12/18	im	Non-neuron
12/19	im iv	Non-neuron
			12/20	im	Non-neuron
12/21	im	Non-neuron
			12/22	im	Non-neuron
12/23	im	Non-neuron
			12/24	im	Non-neuron
12/25	im	Neuron
			12/26	im	Neuron
12/27	iv	Mix
			12/28	im	Neuron

12/29	im	Neuron
			12/30	im	Neuron
12/31	im	Neuron
			1/1	im	Neuron
1/2	im	Mix
			1/4	im	Non-neuron
1/5	iv	Non-neuron
			1/6	Intravenous infusion	Non-neuron
1/7	im	Non-neuron
			1/8	im	Non-neuron

Embodiment 23:

A diagnosis is suffered from DMD, can have the 10 years old boy who walks several steps under the situation of support to treat.He can't independently stand up or independently take a seat in bed.He can lift arm high to 30 ⁰, up to the ancon level.After the some months treatment, his CPK level begins to descend, and he can independently stand up, and a mug water can be lifted high excessive the shower.His body weight does not reduce.

This patient's infusion protocol is referring to table 22.

Table 22

Date	Route of administration	Cell type
			1/20	im	Non-neuron (proof load)
1/23	im	Non-neuron
			1/24	im iv	Non-neuron
1/25	im	Non-neuron
			1/26	im	The neuron non-neuron
1/27	im	Non-neuron
			2/1	im	Non-neuron
2/2	im	The neuron non-neuron
			2/3	im	Non-neuron
2/4	im	The neuron non-neuron
			2/5	im	Neuron
2/6	im	Non-neuron
			2/7	im	Non-neuron
2/8	im	Neuron

		Non-neuron
			2/10	im	The neuron non-neuron
2/11	im	The neuron non-neuron
			2/12	im	The neuron non-neuron
2/14	im iv	The neuron non-neuron
			2/15	im	The neuron non-neuron
2/16	im	The neuron non-neuron
			2/17	im	The neuron non-neuron
2/18	im	The neuron non-neuron
			2/21	im	The neuron non-neuron
2/22	im	The neuron non-neuron
			2/23	im	The neuron non-neuron
2/24	im	Non-neuron
			2/25	im	Neuron
2/26	im	The neuron non-neuron
			2/27	im	The neuron non-neuron
2/28	im	The neuron non-neuron
			3/28	im	The neuron non-neuron
3/29	im	Neuron
			3/30	im	The neuron non-neuron
4/4	im	Neuron
			4/5	im	The neuron non-neuron
4/6	im	Neuron

		Non-neuron
			4/7	im	The neuron non-neuron
4/8	im	The neuron non-neuron
			4/9	im	The neuron non-neuron
5/16	im	The neuron non-neuron
			5/17	im	The neuron non-neuron
5/18	im	The neuron non-neuron
			5/19	The iv infusion	Non-neuron
5/20	The iv infusion	Non-neuron
			5/21	im	The neuron non-neuron
5/22	im	The neuron non-neuron
			5/23	im	The neuron non-neuron
7/27	The iv infusion	Neuron
			7/28	Iv infusion im	The neuron non-neuron
7/29	im	The neuron non-neuron
			7/30	im	The neuron non-neuron
7/31	im	Non-neuron
			8/1	im	Non-neuron
8/24	im	The neuron non-neuron
			8/25	im	The neuron non-neuron
8/26	Iv infusion m	Non-neuron
			8/27	Iv infusion im	Non-neuron
8/28	iv	The neuron non-neuron

8/29	Iv infusion im	Non-neuron
			8/30	im	Non-neuron
8/31	im	Non-neuron
			9/1	im	Non-neuron
9/2	Iv infusion im	Non-neuron
			9/3	Iv infusion im	Non-neuron
9/4	im	Non-neuron
			9/5	im	Non-neuron
11/17	iv im	Mix non-neuron
			11/18	Iv infusion im	The neuron non-neuron
11/19	Iv infusion im	The neuron non-neuron
			11/20	im	Non-neuron
11/22	The iv infusion	Neuron
			11/23	iv im	Mix non-neuron
11/24	iv im	Mix non-neuron
			11/25	im iv	Mix
11/26	im iv	Mix
			11/27	iv im	Non-neuron
11/28	iv	Mix
			11/29	iv	Mix
11/30	im	Mix
			12/1	iv im	Non-neuron
12/2	iv im	Non-neuron
			12/3	iv im	Non-neuron
12/4	iv	Mix
			12/5	im iv	Non-neuron
1/2	im	Mix

1/3	im	Non-neuron
			1/4	im	Non-neuron
1/5	Iv infusion im	Non-neuron
			1/6	Iv infusion im	Mix non-neuron
1/7	The iv infusion	Mix
			1/8	Iv infusion im	Mix non-neuron
1/9	im	Non-neuron

The treatment of heart disease and imbalance

According to the present invention's practice, it is treatment heart disease and imbalance that the another kind of hES cell transplantation is used, and includes but not limited to: restrictive cardiomyopathy, heart failure, sinus bradycardia and coronary heart disease.Certain part noble cells is mixed patient's heart tissue, regenerate and repair impaired muscle.

Though can change dosage regimen to adapt to concrete patient, the typical scenario of treatment heart disease and imbalance is included in carries out intramuscular and intravenous injection every other day in 2 weeks, in ensuing 2 weeks, injected in per 3 days, the 3rd, 6,10 and intramuscular injection in 12 months every month and intravenous infusion once.Can in the bypass surgery process, around affected area, carry out intracardiac injection.

Embodiment 23:

Suggestion suffers from patient's implantable pacemaker of hole sex cords syndrome (Sinus Corda Syndrome) and sinus bradycardia.Put into practice this patient of treatment according to the present invention, by intravenous injection, subcutaneous injection, intramuscular injection or intracardiac injection or during angiography, give about 750,000-160,000,000 hES cell and its derivant, wherein said hES cell and its derivant comprise the hematopoietic stem cell CFU-GM.Sign with this patient behind the hES cell therapy is improved, and does not also need pacemaker.

This patient's infusion protocol is referring to table 22.

Table 22

Date	Route of administration	Cell type
			7/6	Proof load	Non-neuron
7/19	im x 2	Neuron
			7/21	im x 2	Neuron
7/26	im x 2	Neuron
			8/4	im x 2	Neuron

8/10	im x 4	Neuron
			8/26	im x 2	Neuron
9/1	im x 2	Neuron
			9/8	iv	Neuron
9/15	im	Neuron and non-neuron mixture
			9/22	iv im	The neuron non-neuron
9/28	The tail side	Neuron
			10/13	iv x 2	Neuron and non-neuron mixture
10/27	im x 2	Neuron and non-neuron mixture neuron
			11/8	im x 2	Non-neuron
3/23	In the knee joint endoprosthesis	Neuron
			4/14	In the knee joint endoprosthesis	Neuron
5/18	In the knee joint endoprosthesis	Neuron
			6/14	The tail side	Neuron

The treatment of cancerous cell and cancerous tissue

Perhaps, can give hES cell according to the present invention's practice, to replenish cancer patient's conventional chemotherapy.In conventional chemotherapy, give cytotoxic agent with destruction of cancer cells.Yet cytotoxic agent can not be distinguished normal cell and cancerous cell, and may destroy patient's non-cancerous cell, comprises the immune cell of patient.The result is, when carrying out chemotherapy and chemotherapy a period of time after stopping, because compromised immune, the cancer patient is easily infected.By hematopoietic stem cell, neuronal stem cell and hES cell being carried out the patient of chemotherapy, part injection cell will be divided into new immune system, replace by the destructive leukocyte of chemotherapy, erythrocyte, platelet and other cell.Simultaneously, can promote immune system by regulating mitosis mechanism with the hypoplasia bone marrow regeneration that recovery normal cell communication path causes radiation and chemotherapy, and then stop the mitosis of imbalance.Can give hES cell and/or its derivant by intravenous or intramuscular injection, perhaps directly give growth site.These cells also act on unusual hedgehog approach and to its control, to prevent excessive propagation.

Embodiment 24:

The patient who suffers from III phase adenocarcinoma carries out chemotherapy and not success of operative treatment.Because begin to occur the many places hepatic metastases and tuberosity occurs and abdominal cavity and pancreas posterior tubercle, this needs of patients supportive care.

According to the present invention practice, it is about 750 to give this patient, 000-160, and 000,000 hES cell and its derivant cause remarkable improvement, comprise having recovered heterogeneous liver (nodosity before) and the increase of the strong echo of lymph node (echogenic) area of homogeneous.

This patient's infusion protocol is referring to table 23.

Table 23

Date	Route of administration	Cell type
			3/23	Proof load	Non-neuron
3/24	im	Non-neuron
			3/25	im	Non-neuron
3/26	im	Non-neuron
			3/27	im	Non-neuron
3/28	iv	Non-neuron
			3/29	iv	Non-neuron
3/30	iv	Non-neuron
			3/31	iv	Neuron
4/1	iv	Non-neuron
			4/2	im	Non-neuron
4/3	iv	Non-neuron
			4/4	iv	Non-neuron
4/5	iv im x 2	The neuron non-neuron
			4/6	iv im	The non-neuron neuron
4/7	iv	Non-neuron
			4/8	iv	Non-neuron
4/9	iv	Non-neuron
			4/10	iv	Non-neuron
4/11	iv im	The non-neuron neuron
			4/12	iv im	The non-neuron neuron
4/13	iv im	The neuron non-neuron
			4/14	iv	Neuron
4/15	iv im	The non-neuron neuron
			4/16	iv im	The non-neuron neuron
4/17	iv	Non-neuron

4/19	iv im	The non-neuron neuron
			4/20	iv im	The non-neuron neuron
4/21	iv im	The non-neuron neuron
			4/23	The iv infusion	Neuron
4/24	iv	Non-neuron
			4/25	im	Non-neuron
4/26	iv	Non-neuron
			4/27	im	Non-neuron
5/2	iv	Non-neuron
			5/3	iv	Non-neuron
5/6	im iv	The neuron non-neuron
			5/7	im	The neuron non-neuron
5/8	iv	Non-neuron
			5/12	iv	Non-neuron
5/16	im iv	The neuron non-neuron
			5/18	The iv infusion	Non-neuron
5/19	iv	Non-neuron
			5/20	im iv	The neuron non-neuron
5/22	Iv infusion iv x 2	Non-neuron
			5/23	Iv infusion iv x 2	Non-neuron

The treatment of aphthous ulcer/lichen planus

Another application of hES cell and/or its derivant is the ulcer of treatment health mucomembranous surface, as the aphthous ulcer in oral cavity.This cell of intravenous, intramuscular and topical administration.Carry out intramuscular or intravenous injection initial 4 months every days, carried out one time intravenous infusion in every 15-30 days.Carry out also that the part is directly used or use by mixing with gel.

Embodiment 25:

The 65 years old women who is covered with the slough that causes pain and aphthous ulcer on the tongue comes to go to a doctor.She is difficult to feed, swallows and talks.After accepting the hES cell, she makes moderate progress; She can more easily swallow and talk, and can mouth Zhang Degeng is big.She also demonstrates the recovery of ulcer and slough.

The treatment in osteoarthritis, arthritis, tetanic property joint

Another application of hES cell and/or its derivant is treatment osteoarthritis, arthritis and tetanic property joint.Carry out intramuscular injection every day in initial 10 days.Intra-articular injection and Sa Lumeizhuo (salumedrol) are blended 750,000-80,000,000 hES cell, duplicate injection after 1 1/2-3 month.

The treatment of injury of brachial plexus

Another application of hES cell and/or its derivant is the treatment injury of brachial plexus, and wherein ill arm is paralysed.By intramuscular, intravenous route this cell is fed brachial plexus, per 11/2 month repeats once, continues 1 year, perhaps makes moderate progress up to this arm.Also adopt intravenous infusion

Embodiment 26:

The patient is 26 years old male with injury of brachial plexus (Lt.) hands, and in the 7-8, he has lost the function of this hands in the past.Behind stem-cell therapy, his left hand is all more much better than in the past up to ancon and wrist, has better motor capacity.He can mobile wrist, and this hands is weakness no longer as before.He also can upwards lift his arm.

The treatment of reproductive disease

According to the present invention's practice, another application of hES cell transplantation is by recovering the fertility treatment reproductive disease of atrophy of testis, ovarian failure and azoospermia patients.

Embodiment 27:

According to the present invention's practice, comprise the hES cell and its derivatives for treatment azoospermia patients of hematopoietic stem cell CFU-GM by local intramuscular injection, cause producing sperm.In addition, also adopt intravenous and intramuscular injection and be injected directly in the testis, also adopt near subcutaneous injection epididymis.

This patient's infusion protocol is referring to table 24.

Table 24

Date	Route of administration	Cell type
			2/1	Proof load	Non-neuron
8/4	im	Non-neuron
			2/5	im	Non-neuron
8/7	im	Non-neuron
			2/2	im	Neuron and non-neuron mixture
7/27	im	Neuron
			3/6	im	Non-neuron
9/1	im	Neuron
			9/2	im	Neuron
9/6	im	Neuron
			9/7	im	Neuron
9/12	im	Neuron
			9/13	im	Neuron and non-neuron mixture
1/17	iv	Non-neuron
			3/27	im	Non-neuron
4/26	im	Neuron
			5/8	im	Non-neuron
5/25	im	Non-neuron
			8/11	im	Non-neuron
8/17	im	Non-neuron
			9/7	im	Non-neuron

Tissue regeneration

According to the present invention's practice, can give the hES cell, with induced tissue regeneration, include but not limited to anathrepsis, treatment liver cirrhosis and formation neovascularity (neovascularization), it can effectively treat the ulcer of degenerative disease and disunion.

Treatment of diabetes

Another embodiment of the invention is that hES cell and/or its derivant are used for the treatment of diabetes, and wherein said cell comprises the insulin-producing CFU-GM.The risk of diabetics generation heart attack or apoplexy is four times of ordinary people, and the risk of its a plurality of organ degeneration and depletion is also higher, and these organs comprise kidney, eyes, nervous system and general immunity.Thereby recover intravital insulin production treatment diabetes by transplanting insulin-producing hES cell, the patient is reduced the demand of insulin, weak side effect also alleviates to some extent.

Though can change dosage regimen adapting to concrete patient, the typical scenario of treatment diabetes is included in intramuscular and intravenous injection weekly twice in first in middle of the month, the 3rd, 6,11 with injection was once weekly in 12 months.Also can carry out intravenous infusion at 6th month.

Embodiment 28:

This patient is the 70 years old male who suffers from diabetes, and he suffers from the too much nosotoxicosis of keto acid, and he takes 52 units of insulin and oral hypoglycemic.Treat in 6 months, it does not need medicine and insulinize, can keep its blood sugar level in half under the normal diet situation in the previous year.He accepts booster injection.

This patient's infusion protocol is referring to table 25.

Table 25

Date	Route of administration	Cell type
			9/5	Proof load	Non-neuron
9/6	im	Neuron
			9/7	im	Neuron
9/8	im	Non-neuron
			9/9	im	Non-neuron
9/12	iv	Neuron
			9/13	im	Non-neuron
9/19	im	Neuron and non-neuron mixture
			9/22	iv	Non-neuron
9/26	im	Neuron and non-neuron mixture
			9/29	im	Neuron and non-neuron mixture
10/3	im	Neuron and non-neuron mixture
			10/5	im	Non-neuron
10/10	im	Non-neuron
			10/13	iv	Non-neuron
10/17	im	Neuron and non-neuron mixture
			10/20	iv	Non-neuron
10/24	iv	Neuron and non-neuron mixture
			10/31	iv	Neuron and non-neuron mixture
11/3	iv	Neuron and non-neuron mixture
			11/7	im	Non-neuron
11/10	im	Non-neuron
			11/14	iv	Neuron

11/17	im	Non-neuron
			11/21	im	Non-neuron
12/12	iv	Non-neuron
			12/19	iv	Neuron
12/26	iv	Non-neuron
			1/4	im	Non-neuron
8/18	im	Non-neuron
			8/21	im	Non-neuron
8/22	im	Non-neuron
			8/24	im	Non-neuron
8/26	im	Non-neuron

Embodiment 29:

The male suffered from uncontrollable diabetes past three year in 54 years old, with 42 units of insulin and oral hypoglycemic treatment.His fasting serum glucose level is 200mg/dl, and his positive level of postprandial blood sugar is 280mg/dl under medicine control.

After three weeks, he stops insulinize with the hES cell therapy, and has reduced the dosage of blood sugar lowering.He feels very good and vigilance more.

This patient's infusion protocol is referring to table 26.

Table 26

Date	Route of administration	Cell type
			1/18	im	Neuron (proof load)
1/19	im	Non-neuron
			1/20	im	Non-neuron
1/21	im	Mix
			1/22	im	Mix
1/23	im	Mix
			1/24	im	Neuron
1/25	im	Neuron
			1/26	im	Non-neuron
1/27	im	Non-neuron
			1/28	im	Neuron
1/29	im	Neuron
			1/30	im	Neuron
1/31	im	Mix

Embodiment 30:

45 years old male suffers from angina and diabetes.He treats with insulin and oral hypoglycemic, and does angioplasty.

Accept the hES cell therapy after 3 months, he stops insulinize, and reduces the dosage of oral hypoglycemic.His blood glucose and MAR show that blood sugar level has obtained better controlled.

This patient's infusion protocol is referring to table 27.

Table 27

Date	Route of administration	Cell type
			8/21	im	Non-neuron (proof load)
8/22	im	Non-neuron
			8/23	im	Non-neuron
8/24	im	Non-neuron
			8/25	im	Non-neuron
8/28	im	Non-neuron
			8/30	im	Non-neuron
9/1	im	Non-neuron
			9/4	im	Non-neuron
9/6	iv	Non-neuron
			9/8	im	Non-neuron
9/11	im	Non-neuron
			9/13	im	Non-neuron
9/18	im	Non-neuron
			9/20	im	Non-neuron
9/25	im	Non-neuron
			9/28	im	Non-neuron
9/29	im	Non-neuron
			9/30	im	Non-neuron
10/9	im	Non-neuron
			10/11	im	Non-neuron
10/18	im	Non-neuron
			10/23	im	Non-neuron
10/25	im	Non-neuron
			10/31	im	Non-neuron
11/2	im	Non-neuron
			11/08	im	Non-neuron
11/11	The iv infusion	Mix
			11/12	The iv infusion	Mix

11/13	im	Non-neuron
			11/15	im	Non-neuron
11/17	im	Non-neuron
			11/18	The iv infusion	Neuron
11/19	The iv infusion	Neuron
			11/20	im	Non-neuron
11/22	im	Non-neuron
			11/25	The iv infusion	Neuron
11/26	The iv infusion	Neuron
			11/27	im	Non-neuron
11/29	iv im	The neuron non-neuron
			11/30	The iv infusion	Neuron
12/1	The iv infusion	Neuron
			12/10	The iv infusion	Mix
12/11	im	Non-neuron
			12/13	im	Non-neuron
12/16	The iv infusion	Neuron
			12/17	The iv infusion	Neuron
12/18	im	Non-neuron
			12/20	im	Neuron
12/21	im	Neuron
			12/22	iv	Neuron
1/5	im	Non-neuron
			1/6	im iv	Non-neuron
1/8	im	Non-neuron

The treatment of interstitial lung disease

Embodiment 31:

The middle-aged women of suffering from ILD (interstitial lung disease) comes to go to a doctor.She is in disease latter stage, SPO during tranquillization ₂Be 69%.Now, by the hES cell therapy, her disease is no longer made progress.This patient's integral body feels much better, and it is breathed also improvement.This treatment is still continuing.Can hear respiratory murmur during auscultation, her whole lung function shows some improvement.

This patient's infusion protocol is referring to table 28.

Table 28

Date	Route of administration	Cell type
			10/16	im	Non-neuron (proof load)
10/17	im	Non-neuron
			10/18	im	Non-neuron
10/19	Iv epidural spraying	The neuron non-neuron
			10/20	The iv spraying	Neuron
10/21	The iv spraying	Neuron
			10/22	Im iv spraying	The neuron non-neuron
10/23	The iv spraying	Neuron
			10/24	The im spraying	Non-neuron
10/25	Im iv spraying	Mix
			10/26	The iv spraying	Mix
10/27	The iv spraying	Mix
			10/28	The iv spraying	Neuron
10/29	The iv spraying	Neuron
			10/30	The iv spraying	Mix
10/31	The iv spraying	Mix
			11/2	The iv spraying	Mix
11/3	The iv spraying	Mix
			11/4	The iv spraying	Mix
11/5	The iv spraying	Mix

11/6	The iv spraying	Mix
			11/7	The iv spraying	Mix
11/8	The iv spraying	Non-neuron
			11/9	The iv spraying	Mix
11/10	The iv spraying	Non-neuron
			11/11	The iv spraying	Mix
11/12	The iv spraying	Mix
			11/13	The iv spraying	Mix
11/14	The iv spraying	Mix
			11/15	The iv spraying	Neuron
11/16	Im iv spraying	The neuron non-neuron
			11/17	Im iv spraying	Non-neuron
11/18	Im iv spraying	Non-neuron
			11/19	Im iv spraying	Non-neuron
11/20	IM IV spraying	Non-neuron
			11/21	Im iv spraying	Non-neuron
11/22	Im iv spraying	Non-neuron
			11/23	The iv spraying	Neuron

11/24	The iv spraying	Non-neuron
			11/25	The iv spraying	Non-neuron
11/26	The iv spraying	Non-neuron
			11/27	The iv spraying	Non-neuron
11/28	Im iv spraying	Non-neuron
			11/29	Im iv spraying	Non-neuron
11/30	Im iv spraying	Non-neuron
			12/1	The iv spraying	Non-neuron
12/2	The iv spraying	Non-neuron
			12/3	The iv spraying	Non-neuron
12/4	The iv spraying	Non-neuron
			12/5	The iv spraying	Non-neuron
12/6	The iv spraying	Non-neuron
			12/7	The iv spraying	Non-neuron
12/8	The iv spraying	Non-neuron
			12/9	The iv spraying	Neuron
12/10	The iv spraying	Neuron
			12/11	The iv spraying	Non-neuron
12/12	The iv spraying	Non-neuron
			12/13	The iv spraying	Non-neuron

12/14	The iv spraying	Non-neuron
			12/15	The iv spraying	Non-neuron
12/16	The iv spraying	Non-neuron
			12/17	The iv spraying	Non-neuron
12/18	The iv spraying	Non-neuron
			12/19	The IV spraying	Non-neuron
12/20	The iv spraying	Non-neuron
			12/21	The iv spraying	Non-neuron
12/22	The iv spraying	The neuron non-neuron
			12/23	Im iv spraying	The neuron non-neuron
12/24	Im iv spraying	The neuron non-neuron
			12/25	im iv	Mix
12/26	The iv spraying	Mix
			12/27	The iv spraying	Mix
12/28	The iv spraying	Mix
			12/29	im iv	Neuron mixes
12/30	Im iv spraying	Neuron
			12/31	Im iv spraying	Neuron mixes
1/1	Im iv spraying	Mix

1/2	Im iv spraying	Mix
			1/3	Im iv spraying	Non-neuron
1/4	Im iv spraying	Non-neuron
			1/5	Im iv spraying	The neuron non-neuron
1/6	Im iv spraying	Mix non-neuron
			1/7	Im iv spraying	Mix non-neuron
1/8	Im iv spraying	Mix non-neuron
			1/9	Im iv spraying	Mix non-neuron
1/10	Im iv spraying	Mix non-neuron
			1/11	Im iv spraying	Mix non-neuron
1/12	Im iv spraying	Mix non-neuron
			1/13	iv	Mix
1/14	Im iv spraying	Mix non-neuron
			1/15	Im iv spraying	The neuron non-neuron
1/16	Im iv spraying	Mix non-neuron
			1/17	im	Mix

	The iv spraying	Non-neuron
			1/18	IM IV spraying	Mix non-neuron
1/19	Im iv spraying	Mix non-neuron
			1/20	Im iv spraying	Non-neuron
1/21	Im iv spraying	Mix
			1/22	Im iv spraying	Mix
1/23	Im iv spraying	Neuron mixes
			1/24	Im iv spraying	Neuron mixes
1/25	Im iv spraying	Neuron mixes
			1/26	Im iv spraying	Neuron mixes
1/27	Im iv spraying	Neuron mixes
			1/28	Im iv spraying	Neuron mixes
1/29	Im iv spraying	Neuron mixes
			1/30	Im iv spraying	Non-neuron
1/31	Im iv spraying	Non-neuron

New drug development

Another application of hES cell is exploitation and detects new drug.For example, can put into practice according to the present invention and cultivate the hES cell, and as the substrate of testing target spot, model of action, picked-up, metabolism, drainage, toxicity and the safety of new chemical entities, drug candidate and new drug in the medicament research and development.In one embodiment, the present invention relates to the method for test compounds to the effect of hES cell and/or its derivant, it is included in this chemical compound existence and cultivates hES cell and/or its derivant and the effect of definite this chemical compound to this cell that obtains by the inventive method down.

Embodiment 32:

Analyze antibiotic tetracycline and ceftriaxone and patients serum effect to the hES cell of cultivation.These medicines are introduced individually or simultaneously with variable concentrations, and research is to the effect of stem cell.At last, determine the effect of the dosage and the medicine of medicine.

Medicine is sent

Another application of hES cell and/or its derivant is that medicine is carried into the site of damage or disease to carry out local delivery.Can in the presence of medicine, cultivate hES cell and/or its derivant, so that this cell is taken in this medicine.Can will load the cell local delivery to treating the site then, medicine diffuses out and then treats this damage or disease from cell in the treatment site.In another embodiment, if damage or affected areas are not suitable for directly using this medicine (for example, this medicine of direct injection is too big to the damage in this zone), can deliver to this damage or affected areas site in addition with loading cell delivery.Virose medicine when this delivery method can be used systemic delivery to object.Compare with other route of administration, this method also can give the medicine of higher concentration this site.In another embodiment, hES cell and/or its derivant can load one or more medicines, and these medicines can improve the therapeutical effect that cell transplantation itself produces.Non-limitative example comprises the loading cell therapy heart disease that contains antihypertensive, contains the loading cell therapy cancer patient and the loading cell therapy SCI that contains neurotrophic factor of chemotherapeutic.In one embodiment, the present invention relates to medicine is delivered to the method for object, it is included in this medicine and has hES cell and/or its derivant of cultivating the inventive method acquisition down, and wherein said cell is taken in this medicine, give the object site with this cell, medicine is delivered to this site.

In one aspect of the invention, cultural method of the present invention can make hES cell and/or its derivant transplant precursor outer contacting medicine and other activating agent, and relative is directly to give the patient with this medicine and activating agent.Cells in vitro contact medicine helps providing the positive effect (as the neurotrophic effect of valproic acid) of this medicine, the toxicity that produces when avoiding whole body to give this medicine simultaneously.

Spinal cord injury treatment: case research

Produced significant result with hES cell and its derivant clinical treatment SCI.In view of implementing original intention of the present invention and patient is that volunteer and their disease that can't cure is the chronic SCI of ASIA A, therefore surpassed the stage that natural neuranagenesis process may take place, so, do not carry out double blinding or placebo-controlled trial according to doctor's oath.The result of this scheme is provided by the specific embodiment of the case research form that provides below.

Case research 1

Transplant the hES cell and implement the SCI symptom that the present invention can cause suffering from the object of SCI and reverse according to methods described herein.

ReRo 1.3.4./000/220905/ α, declaration can't be used the generation C6-C7 fracture of different medical method treatment and 29 years old object of dislocation.The intermammary region territory of this object does not all have sensation downwards, can not independently take a seat, and does not have the control of bladder or intestinal, can not mobile arm, and the unable or tonicity of both legs.The bilateral decubital ulcer that can't heal has taken place in this object in 3 years.Begin under the following conditions to give hES cell and its derivant according to the present invention's practice.

To contain with physiological saline solution and to have an appointment 750,000-80,000, it is 0.25-1.0ml that the pharmaceutical composition of 000 hES cell and its derivant is diluted to final volume, described hES cell and its derivant comprise hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM, carry out karyotyping then, detect pollution, vigor, and, give forearm by subcutaneous injection then with the standard method counting.Behind five minutes, ten minutes, 15 minutes, 30 minutes, one hour and twenty four hours, observe the anaphylactic shock of this object, the pain of injection site or inflammation, the whole body gargalesthesia is blushed or is generated heat.

This object of subcutaneous initial injection for treating of pharmaceutical composition by being resuspended in the 0.25-1.0ml physiological saline solution, this pharmaceutical composition contains 750,000-80,000,000 hES cell and its derivant, wherein said cell comprises hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM.Also carry the hematopoietic stem cell of the equal number that is resuspended in the 0.25-1.0ml physiological saline solution and the initial injection of neuronal stem cell by intramuscular injection.Be resuspended in 750 of 0.25-1.0ml physiological saline solution by intravenous injection at last, 000-80, the initial injection of 000,000 neuronal stem cell CFU-GM.

To contain 750,000-80,000, the pharmaceutical composition of 000 hES cell and its derivant is resuspended in the 2ml physiological saline solution, further be diluted in the 15-40ml physiological saline solution, for the first time initial injection after 7 days by epidural injection give injury site, below the injury site and more than, thereby put into practice direct treatment SCI according to the present invention, wherein said cell comprises the neuronal stem cell CFU-GM.Behind the initial injection one and a half months, repeat epidural injection after four months and after six months and treat.

Except that epidural gives, also by begin treatment 2 with intrathecal injection is suspended in the 2ml physiological saline solution after 5 months contains 750,000-11,000, this object of medicine composite for curing of 000 hES cell and its derivant, wherein said cell comprises hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM.

In addition, by in continuous three days every day twice epidural injection be resuspended in the 2ml physiological saline solution, further be diluted to final volume 4ml contain 750,000-80,000, this object of medicine composite for curing of 000 hES cell and its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.

This patient's infusion protocol is referring to table 29.

Table 29

Date	Route of administration	Cell type
			8/24	Proof load	Non-neuron
8/25	im x 3	hES
			8/27	im x 2	Neuron
8/28	Spraying is used for decubital ulcer	Neuron
			8/29	im x 4	Neuron
9/6	im x 2	Neuron
			9/8	Epidural	Neuron
9/9	iv	Neuron
			9/10	im	Non-neuron
9/14	im	Neuron and non-neuron mixture
			10/4	In the im sheath	Non-neuron
10/7	Im degree of depth spinal cord injection	Non-neuron
			10/15	im	Neuron
10/21	im	Non-neuron
			10/26	iv	Neuron
11/7	im	Non-neuron
			3/1	im	Neuron

	Degree of depth spinal cord injection x 3
			3/2	im x 2	Neuron
3/3	im x 2	Neuron
			3/5	im	Neuron
3/6	im x 2	Neuron
			3/7	im x 2	Neuron
3/8	Im epidural x2	Neuron
			3/9	Epidural catheter	Neuron
3/10	Epidural catheter	Neuron
			3/11	Epidural catheter	Neuron
3/12	Degree of depth spinal cord injection	Neuron
			3/13	Degree of depth spinal cord injection	Neuron
3/14	im	Neuron
			3/15	im	Neuron
3/16	im	Neuron
			3/17	im	Neuron
3/18	im	Neuron
			3/19	im	Neuron
3/20	im	Neuron
			3/21	im	Neuron
3/22	im	Neuron
			3/23	im	Neuron
3/24	im	Neuron
			3/25	im	Neuron
3/26	im	Neuron
			3/27	im	Neuron
3/28	im	Neuron
			3/29	im	Neuron
3/30	im	Neuron
			3/31	im	Neuron
5/1	im	Neuron
			5/2	im	Neuron
5/3	Epidural catheter	Neuron
			5/4	Epidural catheter	Neuron
5/5	Epidural catheter	Neuron
			5/6	im	Neuron
5/8	im	Non-neuron
			5/12	im	Neuron

The neurological health degree of this object of periodical evaluation behind the begin treatment is observed the mental status and the overall sanitary conditions of this object and is significantly improved after two weeks, shown in table 30.

Table 30

Time	The mental status	Health	Behavior	Cranial nerve
					0 day	Depressed	Difference	Courtesy	Normally
3 days	Depressed	Difference	Courtesy	Normally
					15 days	Nourish hope	Generally	Courtesy	Normally
2 months	Glad	Generally	Courtesy	Normally
					3 months	Nourish hope	Generally	Courtesy	Normally
10 months	The very delight life that looks to the future	Generally	Courtesy	Normally

The typical sign and the symptom of the autonomic nervous system damage that evaluation C6-C7 fracture causes during treating.Shown in table 31, observe all detected parameters and significantly and constantly improve, these parameters comprise ability, sense of touch, consciousness, balanced capacity, the feels pain of sensation deep layer pressure ability, ability, involuntary movement that sensible temperature changes, be in a cold sweat, dizziness, blood pressure, dyspnea, abnormal posture and the ability that independently takes a seat when lying down.

Table 31

Time	Deep layer pressure	Sense of touch	Consciousness	Balanced capacity	Pain	Temperature
							0 day	Between breast	The clavicle lower edge	The clavicle lower edge	Can not take a seat	The clavicle upper limb	The clavicle upper limb
3 days	Umbilicus	Between the arm breast	Forearm between the arm breast	Can not take a seat	Between breast	Between breast
							15 days	Below the umbilicus	Xiphisternum	Xiphisternum and inner arm	Take a seat	Xiphisternum and inner arm	Xiphisternum and inner arm
2 months	Ischial spine	Ischial spine	Xiphisternum and inner arm	Take a seat very well	Ischial spine	Ischial spine
							3 months	The thigh upper limb	Ischial spine	Xiphisternum and inner arm	Take a seat	Ischial spine	Ischial spine
10 months	“	Perineum	Umbilicus	Well take a seat	Perineum	Perineum
							05.06	Perineum	Big midleg	Umbilicus	Well take a seat	Perineum	Perineum

Table 31 (continuing)

Time	Cold sweat	Dizzy	BP	Breathe	Abnormal posture when lying down
						0 day	++++	++++	Fluctuation	Diaphragm is breathed	Can not take a seat
3 days	+++	+++	Fluctuation	Diaphragm is breathed	Can not take a seat
						15 days	++	+	Stable	Normally	Stable taking a seat
2 months	Zero	When taking a seat+	Stable	Normally	Take a seat very well
						3 months	Zero	When taking a seat+	Stable	Normally	Take a seat very well
10 months	Zero	When standing+	Stable	Normally	Take a seat with clamp and walking frame
						05.06	Zero	Zero	Stable	Normally	Take a seat and can go ahead several steps with clamp and walking frame

Bladder is usually relevant with the nerve injury that SCI causes with intestinal dysfunction.This damage causes bladder control, bladder stream and filling of bladder consciousness, intestinal control, intestinal drainage time and intestinal consciousness impaired.In therapeutic process, observe all detected parameters and significantly and constantly improve therapeutic outcome shown in table 32.

Table 32

Time	Involuntary movement	Bladder control	Bladder stream	Consciousness (bladder)	Intestinal control	Evaluation time	Consciousness (intestinal)
								0 day	Zero	Zero	Zero	Zero	Zero	3 hours	Zero
3 days	Zero	Zero	Zero	Zero	Zero	3 hours	Zero
								15 days	Zero	Zero	Zero	Zero	Zero	2.5 hour	Zero
2 months	Zero	Zero	Zero	Zero	Zero	2 hours	+
								3 months	Zero	Zero	Zero	Beginning	+	0.5 hour	++

Assessment changes as the upper part of the body motor capacity of C6-C7 injury site neuranagenesis evidence during treating.Observe head movement, wrist and finger motion, finger strength, tendon reflex, quadruped locomotion intensity, amyotrophy and grip and significantly and constantly improve, shown in table 33.

Table 33

Time	Shoulder L R	Wrist and finger L R	Tendon reflex L R	Strength S FA W F	Atrophy S FA W F	Grip
							0 day	Shrug	The sagging finger of wrist falls into disuse does not have motion	Biceps is (brisk) bilateral fast	4 2 3 0	0 3+4+4+	Zero B/L
3 days	Shrug	The sagging finger of wrist falls into disuse does not have motion	The quick bilateral of biceps	4 2 3 0	0 3+4+4+	Zero B/L
							15 days	Shrug+	The sagging improvement of wrist can be held the removable wrist of thing	Fast	4 3 4 3	0 3 4 4+	There is grip can hold glass
2 months	+	“	Indeterminate (Equivocal)	4 4 4 4	0 2+3+3+	Food can be put into mouth
							3 months	+	Motion is got up much better	Indeterminate	4 4 4 4	0 2+3+3+

The S=shoulder; The FA=forearm; W=hands wrist; The F=hands refers to; A L=left side; The R=right side

Strength: the 0th, poor, the 4th, well

Atrophy: the 0th, well, and the 4th, poor

Assessment changes as the lower part of the body motor function of C6-C7 injury site neuranagenesis evidence during treating, and comprises buttocks motion, knee kinematics, toe movement, tendon reflex, extremity intensity, amyotrophy and vola reaction.Though any parameter of test all not have to improve during studying, treat 3 months after, this object can be stood under the assisting of walking frame, and is shown in table 34.

Table 34

Case research 2

The 22 years old object of paraplegia SCI after the wound takes place in ReRo 1.3.4./001/051004/ α 001 because fall back D6-D7 fracture from the second floor roof.Two talentes that gone into a coma after this object falls recover consciousness, but diplegia takes place, and the lower part of the body from the intermammary region to the foot lost panesthesia and motor capacity, can not lift foot and lower limb, can not independently take a seat or stand up to achieve the sitting posture gesture.The permanent bed of this object, loss of bladder and intestinal control ability rely on household's support fully, but its upper limb is uninfluenced.

According to the present invention's practice, begin to contain the pharmaceutical composition of hES cell and its derivant after 5 months in damage.In 5 months, this object recovers bladder and intestinal control ability, and can take a seat under unsupported condition, slides by mentioning on the buttocks cunning.Sensory perception returns to the perineum level.This object can independently move to self in the walking frame, can be in walking frame skilled standing and do not need knee to support.This treatment causes this object to walk with walking frame under the help of knee brace, and resumes work, that is, he has regained normal life style.

This patient's infusion protocol is referring to table 35.

Table 35

Date	Route of administration	Cell type
			10/5	Proof load	Neuron
8/17	im x 3	Neuron
			8/18	im	Neuron
8/19	im	Neuron
			9/5	im	Non-neuron
9/6	im x 2	Neuron
			9/7	im	Neuron
9/8	im	Neuron
			9/10	im	Non-neuron
9/13	im	Neuron and non-neuron mixture
			9/14	im	Neuron and non-neuron mixture
9/15	im	Non-neuron
			11/6	Epidural catheter	Neuron
11/7	Epidural catheter x 3	Neuron
			11/8	Epidural catheter x 3	Neuron
11/9	Epidural catheter x 3	Neuron
			11/10	Epidural catheter x 3	Neuron
11/11	iv	Neuron
			11/12	im	Neuron
11/13	iv	Neuron
			11/14	iv	Neuron
11/15	iv	Neuron
			11/16	im	Non-neuron
11/18	im	Non-neuron
			1/12	im	Neuron
2/14	im	Non-neuron
			2/15	im	Neuron
2/16	im	Non-neuron

2/17	im	Non-neuron
			2/18	im	Neuron
3/26	im	Neuron
			3/27	Epidural catheter x 3	Neuron
3/28	Epidural catheter x 4	Neuron
			3/29	Epidural catheter x 2	Neuron
3/30	Epidural catheter x 2	Neuron
			3/31	iv	Neuron
5/21	im	Neuron
			5/24	In the sheath	Neuron
5/26	im	Neuron
			7/14	im	Neuron
7/15	im	Neuron
			7/16	im	Neuron
7/17	im	Neuron
			7/18	In the sheath	Neuron
7/19	im	Neuron
			7/21	im	Neuron
7/22	im	Neuron
			7/24	The tail side	Neuron

Case research 3

ReRo 1.3.4./002/040205/ α is because 40 years old object that quadriplegia SCI follows Wry Neck and pain takes place in the C5-C6 damage.This object underwent surgery after six months, then just quadriplegia.Sensation was sunk and dizzy, margin of scapula,superior is felt, lost intestinal and bladder function immediately behind the myasthenia of limbs, operation with top when this object took a seat under the situation of support is arranged.

Perform the operation and begin to contain the pharmaceutical composition of hES cell and its derivant after 9 months according to the present invention's practice.The result of hES cell therapy is, this object can take a seat under the condition that need not to support comfily, moves in the time of can taking a seat in wheelchair and curved to a side, and its lower limb dangle comfily simultaneously.This object significantly improves control above the waist.By motor capacity, independence and active increase, this patient's overall spirit situation is significantly improved.He can have under the condition of support stand, lower limb are strong, can control toe movement, do not have the wrist sagging.According to the improvement of this object situation, the treatment well afoot.

Case research 4

Spinal cord injury and brain injury take place in ReRo 1.3.4./003/260902/ β behind the road traffic accident before 17 years, from then on be limited in 37 years old object on the wheelchair.This object also suffer from the lateral deviation paralysis, can't talk, facial paralysis, memory total loss and uncontrollable bladder.

According to the present invention practice, the pharmaceutical composition that will contain hES cell and its derivant gives this object 1 year 0 two months, and causing can be in the walk with assistance of walking frame, say that several words, cervical region stretch, facial paralysis is eliminated and the memory improvement.

Case research 5

ReRo 1.3.4./004/030505/ α suffers from 56 years old object that the vertebra dislocation (retrovulsion) of fracturing, fracturing after the C5-C8 wound causes contusion of spinal cord and preceding extradural hemorrhage of dependency and paraplegia.This object can not mobile bilateral lower limb, the back acute pain.

According to the present invention practice, contain the pharmaceutical composition 1 year of hES cell and its derivant, cause both legs to recover some strength, recover sensory perception (waving), and can under the help of walking frame, stand and backache does not take place as both legs.This object can walk under the help of walking frame, recovers bladder control and consciousness and overcome cold sweat and dizzy outbreak.

This patient's infusion protocol is referring to table 36.

Table 36

Case research 6

ReRo 1.3.4./005/130705/ β, diagnosis has 25 years old object of rich thatch spondylopathy (Potts Spinedisorder), paraplela inferior to carry out three operations in the D6 level, is the ventromyel decompression.This object be unable to do without wheelchair, can not take a seat under unsupported situation, and leg muscle relaxes and paralyses, and does not have intestinal control, drains when lying down, and does not have bladder sensation.

After damage 11 years 06 months, give the pharmaceutical composition that this object contains hES cell and its derivant according to the present invention's practice.Cause back strength to strengthen, can under unsupported condition, take a seat in 1 year according to this therapeutic scheme, and feel the weight of lower limb.This object has also recovered the backache and the dysmenorrhea of intermenstrual period.Observe the sensation of thigh, lower limb and the recovery of bladder sensation and control.This object can be in the walk with assistance of walking frame, and the motor function recovery of both legs is good.The treatment well afoot.

This patient's infusion protocol is referring to table 37.

Table 37

Date	Route of administration	Cell type
			7/13	Proof load	Non-neuron
7/27	im	Neuron
			7/28	im	Non-neuron
7/29	im	Neuron
			8/3	im x 2	Neuron
8/4	im x 2	Non-neuron
			8/5	im	Neuron
8/9	im	Neuron
			8/12	im x 2	Neuron
8/16	im	Neuron
			8/23	im x 2	The hES neuron
8/30	im x 2	Neuron
			9/1	im x 4	Neuron
9/2	im x 2	Neuron
			9/8	iv	Neuron
9/12	im	Neuron
			9/14	im	Neuron and non-neuron mixture
9/22	iv	Neuron
			9/27	im	Neuron and non-neuron mixture
9/29	im	Neuron and non-neuron mixture
			9/30	im	Neuron
10/4	im	Neuron and non-neuron mixture

10/6	Epidural x 2 bottles	Neuron
			10/10	im	Neuron
10/19	im	Non-neuron
			10/21	im	Neuron
10/24	Epidural	Neuron
			11/7	Epidural	Neuron
11/14	iv	Neuron
			11/15	im	Non-neuron
11/18	im	Non-neuron
			12/1	In the sheath	Neuron and non-neuron mixture
12/7	Degree of depth spinal cord injection	Non-neuron
			12/23	im	Neuron
12/26	im	Non-neuron
			1/5	im	Neuron
1/11	im	Neuron
			1/17	im	Neuron
1/24	iv	Neuron
			1/26	im	Neuron
2/1	im	Neuron
			2/3	im	Neuron
2/9	im	Non-neuron
			2/15	im	Non-neuron
2/22	im	Neuron
			3/1	im	Non-neuron
3/9	im	Non-neuron
			3/21	im	Neuron
3/28	im	Neuron
			4/13	Epidural catheter	Neuron
4/26	im	Neuron
			5/2	im	Non-neuron
5/4	Epidural catheter	Neuron
			5/5	Epidural catheter	Neuron
5/6	Epidural catheter	Neuron
			5/7	im	Neuron
5/21	im	Neuron
			6/5	im	Neuron
6/12	im	Neuron
			6/26	iv x 2	Non-neuron
7/8	im	Neuron

Case research 7

ReRo 1.3.4./006/220805/ α, 30 years old object suffering from C6-C7 paraplegia SCI can not mobile lower limb, do not have intestinal control and intestinal to feel.This object has only the intermammary region zone that makes progress that clear and deep layer pressure sensation is arranged.Therefore, this object is difficult to take a seat.The hand-power gas of this object is very little, and a little less than the finger motion very, this object is difficult to breathe.

According to the present invention's practice, by contain this object of medicine composite for curing of hES cell and its derivant after about 3 months in damage.But this object recover under no supporting condition the ability that takes a seat, no longer dizzy perception groin pressure, can feel that if ancon inboard patient attempts the mobile shank pain of can feeling.The breathing easily of this object, bladder and intestinal is felt, shank is felt can be stood several minutes having under the situation of support now.The finger strength and the motor capacity of this object also have raising.The treatment well afoot.

This patient's infusion protocol is referring to table 38.

Table 38

Date	Route of administration	Cell type
			2/22	Proof load	Non-neuron
2/23	im	Neuron
			2/24	im	Neuron
2/27	im	Non-neuron
			2/28	im	Non-neuron
3/2	im	Neuron
			3/3	im	Neuron
3/8	Epidural catheter	Neuron
			3/9	Epidural catheter x 2	Neuron
3/10	Epidural catheter x 2	Neuron
			3/11	iv	Neuron
3/20	im	Neuron
			3/22	im	Neuron
3/23	im x 2	Neuron
			3/24	im	Non-neuron
3/27	im	Non-neuron
			4/5	In the sheath	Neuron
4/12	im	Neuron
			5/1	Im epidural catheter x 4	Neuron

5/2	The im epidural catheter	Neuron
			5/10	Epidural catheter x 4	Neuron
5/11	Epidural catheter x 4	Neuron
			5/12	Epidural catheter x 4	Neuron

Case research 8

ReRo 1.3.4./007/221005/ β because 26 years old object of D6SCI and paraplegia can not mobile both legs, but can take a seat under unsupported situation.This object does not have bladder or intestinal control, has only top, intermammary region territory to feel.

After damaging about 10 months, give the pharmaceutical composition that this object contains hES cell and its derivant according to the present invention's practice.This object recovers the health side up to umbilicus zone (unbiliousregion), and bilateral axil territory is to the sensation of hipbone.This object can be in the walk with assistance of walking frame and clamp, and both legs have motor capacity.

This patient's infusion protocol is referring to table 39.

Table 39

Date	Route of administration	Cell type
			9/26	Proof load im	Neuron
9/27	im	Neuron and non-neuron mixture
			9/28	im	Neuron and non-neuron mixture
9/29	im	Neuron
			9/30	im	Neuron and non-neuron mixture
10/1	im	Neuron and non-neuron mixture
			10/3	im	Neuron and non-neuron mixture
10/4	In the im sheath	Non-neuron
			10/5	m	Neuron
10/6	Epidural	Neuron
			10/8	m	Non-neuron
10/10	im	Neuron
			10/12	Im degree of depth spinal cord injection	Neuron
10/15	iv	Neuron
			10/19	im	Non-neuron
10/24	im	Neuron and non-neuron mixture
			10/25	Epidural	Neuron

12/8	Degree of depth spinal cord injection	Non-neuron
			12/10	In the sheath	Non-neuron
12/11	im	Non-neuron
			12/12	Degree of depth spinal cord injection	Non-neuron
12/13	im	Neuron
			12/14	Epidural	Neuron
12/15	im	Neuron
			2/13	im	Neuron
2/15	Epidural	Neuron
			2/16	iv	Neuron
2/17	im	Neuron
			5/24	im iv	Neuron
5/25	im iv	The non-neuron neuron
			5/26	im iv x 2	The neuron non-neuron, neuron
5/27	im x 2 iv x 2	The non-neuron neuron
			5/28	im	Neuron, non-neuron
5/29	im	Neuron, non-neuron
			5/30	The epidural infusion	Neuron
5/31	im	Neuron

Case research 9

ReRo 1.3.4./008/151005/ β suffers from the traumatic quadriplegia paralysis and is difficult to talk and breathing, 27 years old stiff object of cervical region.The paralysis of the lower limb of this object, this object can't mobile fingers, health remainder numbness.

After damaging about 2 years, beginning is treated according to the pharmaceutical composition that the present invention's practice contains hES cell and its derivant.The neck movement of this object improves, talk easily, the control ability of tone is improved.This object finds to breathe not difficulty in the past, and motor function is recovered to some extent, can do some finger motions.The spasm extent of lower limb alleviates, and this object can independently take a seat.Also recovered toe movement, this object can mobile shoulder.

This patient's infusion protocol is referring to table 40.

Table 40

Date	Route of administration	Cell type
			5/26	Proof load	Non-neuron
5/27	im	Neuron
			5/28	im	Neuron
5/29	im	Neuron
			5/30	im	Neuron
5/31	im	Neuron
			6/1	im	Neuron
6/8	im x 2	The neuron non-neuron
			6/9	im	Neuron
6/10	im x 2	The neuron non-neuron
			6/11	im x 2	The neuron non-neuron
6/12	im x 2	The neuron non-neuron
			6/13	im x 2	The neuron non-neuron
6/14	In the sheath	Neuron
			6/15	im x 2	The neuron non-neuron
6/16	im x 2	The neuron non-neuron
			6/17	im x 2	The neuron non-neuron
6/18	im	Non-neuron
			6/19	im x 2	The neuron non-neuron
6/20	im x 2	Neuron
			6/21	im x 2	The neuron non-neuron
6/22	im x 2	Neuron
			6/23	iv x 2	Non-neuron
6/24	iv x 2	Non-neuron
			6/25	iv x 2	Non-neuron
6/26	Epidural catheter x 2	Neuron
			6/27	Epidural catheter x 2	Neuron
6/28	Epidural catheter x 3	Neuron

7/21	im iv	The neuron non-neuron
			7/22	im x 2	The neuron non-neuron
7/23	im x 2	Neuron
			8/22	In the sheath	Neuron

Case research 10

ReRo 1.3.4./009/300106/ α, object was once gone through vehicle accident in 25 years old, can not take a seat under unsupported situation, and vertebra tends to bending, and this object has been lost bladder and intestinal control, and the lower limb of this object does not have strength fully.

After damaging about 2 years, put into practice by containing this object of medicine composite for curing of hES cell and its derivant according to the present invention.This object demonstrates quick improvement, and can take a seat under unsupported situation.Dizziness, both feet do not take place this object can move, recover the finger tip sensation, and can feel cold flow (chill flowing through the spine) by vertebra.

Case research 11

ReRo 1.3.4./010/020206/ β follows knee to shrink because traffic accident can not be stood at 26 years old object of D12-L1 generation SCI.This object can take a seat under unsupported situation, and the extremity sensation is normal.This object does not have bladder or intestinal control ability, and spasm extent improves.

After damaging about 12 years, contain the pharmaceutical composition of hES cell and its derivant to treat this object according to the present invention's practice.The leg exercise function of this object is obviously recovered, and spasm extent reduces, and its right lower limb can stretch fully, and left leg strength amount is restored.This object has recovered the auxiliary ability of standing and walking down at clamp and walking frame.This object has also recovered bladder and intestinal sensation.

This patient's infusion protocol is referring to table 41.

Table 41

Date	Route of administration	Cell type
			3/27	Proof load	Non-neuron
3/29	im iv	The non-neuron neuron
			3/30	im	Neuron
3/31	im	Neuron
			4/1	im	Neuron
4/2	im	Non-neuron
			4/3	im	Neuron

4/4	im	Neuron
			4/5	The tail side	Neuron
4/7	Epidural	Neuron
			4/8	im	Neuron
4/10	im	Neuron
			4/13	im	Neuron
4/14	im	Neuron
			4/14	im	Neuron
4/16	im	Neuron
			4/18	iv	Non-neuron
4/22	im	Neuron
			4/23	im	Neuron
4/24	Epidural catheter	Neuron
			4/25	Epidural catheter	Neuron
4/26	iv	Neuron
			4/27	im	Neuron
4/28	im	Neuron
			4/29	im	Neuron
4/30	im	Neuron
			5/1	im	Neuron
5/2	im	Non-neuron
			5/3	im	Neuron
5/4	im	Neuron
			5/5	im	Neuron
5/7	im	Neuron
			5/8	im	Non-neuron
5/9	im	Non-neuron
			5/10	im	Neuron
5/11	The tail side	Neuron
			5/12	im	Neuron
5/13	im	Neuron
			5/14	im	Neuron
5/16	im	Non-neuron
			5/17	im	Neuron
5/20	Epidural	Neuron
			5/21	im	Neuron
5/22	im	Neuron
			5/23	im	Neuron
5/24	im	Neuron

Case research 12

This patient is 26 years old male that paraplegia takes place in the damage back between the D6-D8.He causes wound in JIUYUE, 2004 generation vehicle accident.He is unable to leave the bed fully, and the following not sensation of chest does not have the control of bladder or intestinal, chest following not sensation or motor capacity.The grow decubital ulcer of profound degree of back can be seen rumpbone.

This patient is at beginning on April 27th, 2006 hES cell therapy.Because he can't carry out any O/T program decubital ulcer, give the cell of doses by intravenous and intramuscular every day, and the hES cell directly is applied to his decubital ulcer the most at last.Also give cell by intravenous infusion.

At the 11st day of treatment, he can stand under the help of complete clamp (waist is to ankle) and walking frame and walk out for 2 steps.As time passes, his muscle strength strengthens, and he can walk out nearly 100 steps after 4 months, and can stand and reach 20 minutes.He can the perception shank, also can move.He can feel that also bladder and intestinal are full, can only walk by the support of knee brace and walking frame.His decubital ulcer heals, and continues his research.

This patient's infusion protocol is referring to table 42.

Table 42

Date	Route of administration	Cell type
			4/28	im iv	Neuron (proof load)
4/29	iv im	The neuron non-neuron
			4/30	iv im	The neuron non-neuron
5/1	iv im	The neuron non-neuron
			5/2	iv im	The neuron non-neuron
5/3	Iv infusion im	The neuron non-neuron
			5/4	Iv infusion im	The neuron non-neuron
5/5	iv im	The neuron non-neuron
			5/6	iv im	The neuron non-neuron

5/8	Iv infusion im	Neuron
			5/9	Iv infusion im	The neuron non-neuron
5/10	Iv infusion im	The neuron non-neuron
			5/11	iv im	Neuron
5/12	iv im	The neuron non-neuron
			5/13	iv im	Neuron
5/14	iv im	The neuron non-neuron
			5/15	Iv infusion im	Non-neuron
5/16	Iv infusion im	Non-neuron
			5/17	iv im	The neuron non-neuron
5/18	iv im	The neuron non-neuron
			5/19	iv im	The neuron non-neuron
5/20	iv im	The neuron non-neuron
			5/21	iv im	The neuron non-neuron
5/22	Iv infusion im	Non-neuron
			5/23	Iv infusion im	Non-neuron
5/24	iv im	The neuron non-neuron
			5/25	iv im	The neuron non-neuron
5/26	iv im	The neuron non-neuron
			5/27	iv im	The neuron non-neuron
5/28	iv	Neuron

	im	Non-neuron
			5/29	Iv infusion im	The neuron non-neuron
5/30	Iv infusion im	The neuron non-neuron
			5/31	iv im	The neuron non-neuron
6/1	iv im	The neuron non-neuron
			6/2	iv im	The neuron non-neuron
6/3	iv im	The neuron non-neuron
			6/4	iv im	The neuron non-neuron
6/5	Iv infusion im	The neuron non-neuron
			6/6	Iv infusion im	The neuron non-neuron
6/7	iv im	The neuron non-neuron
			6/8	im iv	The neuron non-neuron
6/9	im iv	The neuron non-neuron
			6/10	im iv	The neuron non-neuron
6/11	im iv	The neuron non-neuron
			6/12	The iv infusion	Neuron
6/13	Iv infusion im	Non-neuron
			6/14	iv im	The neuron non-neuron
6/15	iv im	The neuron non-neuron
			6/16	iv im	The neuron non-neuron
6/17	iv im	The neuron non-neuron

6/18	iv im	The neuron non-neuron
			6/20	Iv iv infusion im	Neuron
6/21	iv	The neuron non-neuron
			6/22	iv im	Non-neuron (2)
6/23	iv	Non-neuron (2)
			6/24	iv	Non-neuron (2)
6/25	iv	Non-neuron (2)
			6/26	Iv iv infusion	Non-neuron
6/27	Iv iv infusion im	Non-neuron
			6/28	iv im	Neuron
6/29	iv im	Neuron non-neuron x 2
			6/30	iv im	Neuron non-neuron x 2
7/1	iv im	Neuron non-neuron x 2
			7/2	iv im	Neuron non-neuron x 2
7/3	Iv iv infusion im	Neuron non-neuron x 2
			7/4	Iv iv infusion im	The neuron non-neuron
7/5	iv im	Neuron x 2
			7/6	iv im	Neuron x 2
7/7	iv im	Neuron
			7/8	iv im	Neuron non-neuron x 2
7/9	iv im	Neuron

		Non-neuron
			7/11	Iv iv infusion im	The neuron non-neuron
7/12	Iv iv infusion	Neuron
			7/17	The im dressing	The neuron non-neuron
7/18	Iv infusion dressing	Non-neuron
			7/19	Iv infusion im	Non-neuron
7/20	Iv im dressing	The neuron non-neuron
			7/21	Iv im dressing	The neuron non-neuron
7/22	Iv im dressing	The neuron non-neuron
			7/23	iv im	The neuron non-neuron
7/24	iv im	The neuron non-neuron
			7/25	The im dressing	Neuron non-neuron x2
7/26	The m dressing	Neuron non-neuron x 2
			7/27	im	The neuron non-neuron
7/28	The im dressing	Non-neuron
			7/29	The im dressing	The neuron non-neuron
7/30	im	The neuron non-neuron
			7/31	The im dressing	Non-neuron
8/1	The im dressing	Non-neuron x 2
			8/2	im	Non-neuron x 2

	Dressing
			8/3	im	Non-neuron x 2
8/4	im	Non-neuron x 2
			8/5	The im dressing	Non-neuron x 2
8/6	im	Non-neuron x 2
			8/7	The im dressing	Non-neuron x 2
8/8	im	Non-neuron x 2
			8/9	The im dressing	Non-neuron x 2
8/10	im	Non-neuron x 2
			8/11	im	Non-neuron x 2
8/12	The im dressing	Non-neuron x 2
			8/13	im	Non-neuron x 2
8/14	The im dressing	Non-neuron x 2
			8/15	The im dressing	Non-neuron x 2
8/16	im	Non-neuron x 2
			8/17	im	Non-neuron x 2
8/18	Iv infusion im	Neuron x 3
			8/19	The im dressing	Neuron x 3
12/5	im	Non-neuron
			12/6	The iv infusion	Non-neuron
12/7	Iv infusion im dressing	Non-neuron
			12/8	Epidural (in the sheath)	Neuron
12/9	The im dressing	Non-neuron x 2
			12/10	The im dressing	Non-neuron x 2
12/11	The im dressing	Non-neuron x 2
			12/12	Epidural (conduit)	Neuron x 2
12/13	Epidural (conduit)	Neuron x 2
			12/14	Epidural (conduit)	Neuron x 2

12/15	Iv infusion im	Neuron, non-neuron
			12/16	The iv infusion	Neuron, non-neuron

Case research 13

This patient is the accident of riding in June, 2006,22 years old male of T-12, L-1 paraplegia.His waist is following unable, does not have the control of intestinal or bladder, the following numbness of waist.

This patient begins to accept the treatment in six weeks by a definite date in JIUYUE, 2006.He relies on complete clamp (waist is to ankle) and walking frame can stand and several steps of walking in week to the hES cellular response.He makes progress, and can stand by knee brace and walking frame to be thirty minutes long.He has sensation and intestinal and bladder control completely now.He can keep good balance with walking stick and crutch.He can rely on clamp and crutch stair activity.

This patient's infusion protocol is referring to table 43.

Table 43

Date	Route of administration	Cell type
			10/4	im	Non-neuron (proof load)
10/5	im	Non-neuron
			10/6	im	Non-neuron
10/7	im	Non-neuron
			10/8	im	Non-neuron
10/9	The iv infusion	Neuron
			10/10	The iv infusion	Neuron
10/11	im	Non-neuron
			10/12	im	Non-neuron
10/13	im	Non-neuron
			10/14	im	Non-neuron
10/15	im	Non-neuron
			10/16	The tail side	Neuron
10/17	im	Non-neuron
			10/18	im	Non-neuron
10/19	Iv infusion im	Non-neuron
			10/20	Iv infusion im	Non-neuron
10/21	im	Non-neuron
			10/22	im	Non-neuron
10/23	Lumbar vertebra	Neuron

10/27	im	Non-neuron
			10/25	im	Non-neuron
10/26	im	Non-neuron
			10/27	im	Non-neuron
10/28	im	Non-neuron
			10/29	im	Non-neuron
10/30	Epidural catheter	Neuron
			10/31	Epidural catheter	Neuron
11/1	Epidural catheter	Neuron
			11/2	im	Non-neuron
11/3	im	Non-neuron
			11/4	im	Non-neuron
11/5	im	Non-neuron
			11/6	im	Non-neuron
11/7	im	Non-neuron
			11/8	im	Non-neuron
11/9	The tail side	Neuron
			11/10	im	Non-neuron
11/11	im	Non-neuron
			11/12	im	Non-neuron
11/13	Iv infusion im	Mix non-neuron
			11/14	Im iv infusion	Non-neuron
11/15	im	Non-neuron
			12/31	iv	Mix
1/10	im	Non-neuron
			1/11	The iv infusion	Mix
1/12	The iv infusion	Mix
			1/13	im	Non-neuron
1/14	im	Non-neuron
			1/15	The tail side	Neuron
1/16	im	Non-neuron
			1/17	im	Non-neuron
1/18	im	Non-neuron
			1/19	Lumbar vertebra	Neuron
1/20	im	Non-neuron mixes
			1/21	im	Mix
1/22	im	Mix

1/23	Epidural catheter	Neuron
			1/24	im iv	Neuron
1/25	im	Neuron
			1/26	Infusion im	Mixed nerve unit
1/27	Infusion im	Mixed nerve unit

Case research 14

The patient is because 26 years old women of traumatic paralysis takes place at T-12, L-1 the road traffic accident of in JIUYUE, 2004 generation.The following numbness of her chest does not have intestinal or bladder control ability.Her not motion strength of lower limb be unable to do without wheelchair.

This patient begins the hES cell therapy in March, 2006.She relies on complete clamp (waist is to ankle) and walking frame can stand and several steps of walking after 9 days, and continues to improve.Her current state is: can rely on clamp and walking frame continuous walking, also can rely on knee brace to be stood.Intestinal and bladder sensation recover, and control ability is arranged, and do not re-use catheterization or suppository.Sensation has been improved to the ankle level.

This patient's infusion protocol is referring to table 44.

Table 44

Date	Route of administration	Cell type
			3/27	im	Non-neuron (proof load)
3/29	iv im	The neuron non-neuron
			3/30	im	Neuron
3/31	im	Neuron
			4/1	im	Neuron
4/3	im	Neuron
			4/4	im	Neuron
4/5	Epidural (tail side)	Neuron x 2
			4/7	Epidural (in the sheath)	Neuron x 2
4/9	im	Neuron
			4/10	im	Neuron
4/12	im	Neuron
			4/13	im	Neuron
4/14	im	Neuron

4/15	im	Neuron
			4/16	im	Neuron
4/17	im	Neuron
			4/18	iv	Non-neuron
4/20	im	Neuron
			4/21	im	Neuron
4/22	im	Neuron
			4/24	Epidural (conduit)	Neuron x4
4/25	Epidural (conduit)	Neuron x4
			4/26	Iv (conduit)	Neuron x4
4/27	im	Neuron
			4/28	im	Neuron
4/29	im	Neuron
			4/30	im	Neuron
5/1	im	Neuron
			5/2	im	Non-neuron
5/3	im	Neuron
			5/4	im	Neuron
5/5	im	Neuron
			5/7	im	Neuron
5/8	im	Non-neuron
			5/10	im	Neuron
5/11	EPI (tail side)	Neuron
			5/13	IM	Neuron
5/14	IM	Neuron
			5/16	IM	Non-neuron
5/17	IM	Neuron
			5/18	EPI (conduit)	Neuron x 2
5/19	EPI (conduit)	Neuron x 2
			5/20	EPI (conduit)	Neuron x 2
5/21	im	Neuron
			5/22	im	Neuron
5/23	im	Neuron
			5/24	im	Neuron
5/25	im	Neuron
			10/25	im	Non-neuron mixes
10/26	Im iv infusion	Non-neuron mixes
			10/27	im	Non-neuron

10/28	im	Non-neuron
			10/29	im	Non-neuron
10/30	The tail side	Neuron
			10/31	im	Non-neuron
11/1	im	Non-neuron x 2
			11/2	im	Non-neuron x 2
11/3	Lumbar vertebra	Neuron
			11/4	Infusion	Neuron
11/5	im	Non-neuron x 2
			11/6	im	Non-neuron x 2
11/7	im	Non-neuron x 2
			11/8	Epidural catheter	Neuron
11/9	Epidural catheter	Neuron
			11/10	Epidural catheter	Neuron
11/11	im	Non-neuron x 2
			11/12	The im infusion	Non-neuron mixes
11/13	Tail side infusion	Neuron mixes
			11/14	im	Neuron

HESC's cultural method

HES as the cell line parent material of exploitation according to the present invention is cell-derived from the prenidatory 2-7 in uterus day instar embryo, as 2-4 day instar embryo, as instar embryo on the 3rd.

In the embryo separated, ovum was collected from the people's donor that carries out the conventional IVF cycle of signing Informed Consent Form.Make this ovum fertilization, cultivate with conventional method and obtain the embryo.Extra embryo is cultivated different time, to develop into cell line.

This embryo is suspended in a small amount of minimum essential medium (as RPMI, as containing the RPMI 1640 of 2.2g/L sodium bicarbonate),, separates hES cell among this embryo as concussion by mechanical means.Extra culture medium and progestogen and β hCG agonist are added in the isolated cells.In one embodiment, add progesterone (16-64 μ l 250mg/ml) and β hCG (16-64 μ l 5000iu/ml).The culture of isolated cell was 12-48 hour in the environment of 34-38 ℃ temperature, 3.5-6% carbon dioxide, as 24 hours.

In one embodiment, under anoxia or obvious anoxybiotic condition, cultivate the hES cell, so as when to prevent to break up amplifying cells.The obvious anoxia ＂ of ＂ is defined as being less than about 10%O ₂, as be less than 8%, 6%, 4%, 2% or 1%O ₂Available many gas (CO ₂, O ₂, N ₂) cell culture incubator generation hypoxia condition, wherein by oxygen level being set at 0-20% with the air in the nitrogen replacement culture environment.The example of many gas incubator comprises Fisher 11-730 series, Napco 7000 series, Sanyo MCO series, Jouan IG750 and Heraeus Heracell 150.In another embodiment, at CO ₂Cultured cell in the incubator, the shape and the Position Control oxygen level of the culture bottle by containing cell.For example, when Tissue Culture Flask is vertically placed, the obvious anoxia of many culture medium in this culture bottle.On the contrary, when the Tissue Culture Flask horizontal positioned, the obvious aerobic of this culture medium.Culture medium content (for example head space amount) is in obvious anoxia with obviously change the oxygen content of culture medium between the aerobic in shape that can be by changing Tissue Culture Flask in the incubator and/or position or the culture bottle.

In the amplification stage, make culture bottle keep the upright position, but not horizontal level.In one embodiment, volume is almost completely occupied by culture medium and keeps cultivating in the vertical culture vessel.The result is that most of cell can be not adherent on the culture bottle wall, the obvious anoxia of culture medium.

Under this growth conditions, cell multiplication or proliferating cycle are maximum, obviously suppress cell differentiation procedure.Be 12-48 hour cell generation cycle in the amplification procedure, as 24 hours.

In the going down to posterity or cultivate of hES cell, centrifugal this cell suspension is resuspended in cell precipitation in the fresh culture that contains progesterone and β hCG on a small quantity, and this cell of five equilibrium adds the fresh culture that does not contain progesterone and β hCG.In one embodiment, the ratio of cultivation stem cell equal portions and fresh cell culture medium is about 1:3.5-1:35 volume/volume.In the 34-38 that is filled with the 3.5-6% carbon dioxide ℃ of water leg incubator, under obvious anoxybiotic condition, cultivated this cell once more 12-48 hour, as 24 hours.At this moment, this cell that can go down to posterity perhaps stores stand-by to continue amplification.

By alkaline phosphatase activities with do not exist the characteristic label of cell specification or differentiation to prove, the hES cell of cultivating under this class condition remains undifferentiated substantially form.Yet, notice to obtain the cell mass that breaks up fully or do not break up fully.This cell mass is the mixture of differentiation and undifferentiated cell system, and the ratio of undifferentiated cell and noble cells is about 4:1-10:1.

In order to store the hES cell of amplification, for example be stored in cryogenic refrigerator or the liquid nitrogen, can include but not limited to that 0.2-2% (w/v) dimethyl sulfoxine (DMSO) adds in this culture medium with frozen dose.Used DMSO level is less than frozen used mean level (as 2-84 μ l/0.5ml test tube), with differentiation or the damage of avoiding inducing cell.Frozen agent content can be about 1:500-16:1000 with the ratio that contains the culture medium of stem cell.Other frozen dose includes but not limited to: glycerol, propylene glycol, butanediol, ethanol, glucose, D-glucose, sucrose, trehalose, mannitol, the Barbara Qin (paparavine), Methanamide, probacol (probuchol), curcumin, polyvinylpyrrolidone, Polyethylene Glycol, chondroitin sulfate, mucopolysaccharide, dimethyl sulfoxine, glutamine and Sodium Pyruvate.The frozen temperature of cell suspension can be-15 to-80 ℃ approximately, according to appointment-15 to-40 ℃.Opposite with other technology that stores human embryo stem cell and its derivant, do not need hurried freezing or be stored in " suction pipe (straw) ".After the storage, can be resuspended in fresh culture, prepare to make this cell amplification or differentiation continuously by centrifugal collecting cell with cell precipitation.

Centrifugal (as the centrifugal 5-12 of 700-1400rpm minute) cell suspension is with sedimentation cell, remove and abandoning supernatant, this cell is resuspended in the fresh culture (as RPMI) that contains progestogen and β hCG agonist on a small quantity, thus the hES cell of amplification is partly broken up (for example behind the hES of the fresh separated cell amplification or before expanded cells store the back).The equal portions isolated cell adds the extra culture medium (as minimum essential medium such as RPMI or DMEM, do not have glutamic acid DMEM or contain the 4.5g/L glucose and the DMEM of 3.7g/L sodium bicarbonate as 1X) that does not contain progestogen or β hCG then.Condition (for example in the culture bottle in horizontal positioned) at obvious aerobic was descended cultured cell 12-48 hour, as 24 hours.The obvious aerobic ＂ of ＂ is defined as containing at least about 15%O ₂, as at least about 18% or 20%O ₂Under obvious aerobic conditions, the hES cell begins differentiation.The differentiation pathway of hES cell depends on the culture medium that this differential period is used.In order to produce neuron progenitor cell, cultivate the hES cell with DMEM or its equivalent.In order to produce neuron progenitor cell CFU-GM in addition, cultivate the hES cell with RPMI or its equivalent.After 12-48 hour proliferating cycle, collect this cell, be resuspended in the fresh culture so that continue differentiation.Usually, differential period should not surpass 48-72 hour, can produce the cell that is not suitable for the inventive method transplanting because surpass the undue differentiation of this time.

In case after cell partly breaks up, add frozen dose as mentioned above, cell is stored in-15 to-80 ℃, so that cell is stored stand-by, perhaps preparation is used for the cell of the inventive method.In order to prepare the cell of use, the equal portions isolated cell adds fresh culture (as required, as DMEM or RPMI), cultivates this cell 12-48 hour under obvious anoxybiotic condition, as 24 hours, to prevent further differentiation.Then cell equal portions branch is gone in the fresh culture, continue amplification, or by centrifugal collection and be resuspended in biocompatible solution (as saline) and prepare to transplant.At this moment, cell transplantation can be gone into the patient, or be stored in+4 ℃ to-80 ℃ so that transplant in the future.Can store in any suitable vessel that helps transplanting with clinical use, these containers include but not limited to: test tube, bottle, syringe etc.In one embodiment, promptly to store the cell (for example being stored in the pre-filled syringe) that is resuspended in biocompatible solution with form.When needing, allow the cell equal portions thaw naturally, do not need to thaw with water-bath or incubator.

Under the described storage requirement in biocompatible solution, observe storage and thaw cell viability more still after 6 months 40%, do not detect genotype and change (genetic instability) or phenotypic alternation, as aneuploid or heteroploid.

In each stage of amplification, differentiation and storage, detect the vigor of cell equal portions by trypan blue eliminating and sediments microscope inspection.Also detect the microbial contamination of cell culture equal portions.Other cell suspension equal portions also can be placed on the hemocytometer by sediments microscope inspection, to determine cell density.Also get other equal portions test alkaline phosphatase activities, it is the label of cell culture differentiation state.

Between the storage life, can measure the caryogram of a cell in every month, to detect the genetic instability that causes by cultural method.

Embodiment

Embodiment 1

In the embryo separates, by the carrying out of signature Informed Consent Form people's donor recovery of ova in conventional IVF cycle, this ovum produces 8 follicles.Make this ovum fertilization, cultivate with conventional method and obtain the embryo.Three embryos are implanted in the donor body.Extra embryo is cultivated different time, to develop into hES cell line.

Complete or broken embryo is resuspended in the culture medium.In addition, add 84 μ l progesterone (250mg/ml) and 84 μ l β hCG (5000iu/ml).Detection contains the pollution condition of the culture medium of embryonic cell, discards infected embryo.Increase and store with the embryonic cell of this form.

Cultivate after 1 day, the culture medium that 1ml is contained embryonic cell adds in the 50ml container and contains in the 40ml cell culture medium (DMEM or RPMI) of progesterone and β hCG.In room temperature, contain in the environment of carbon dioxide level and cultivate this culture medium and stem cell.Table 45 shows some experiment conditions that this kind method is followed.

Cultivate after 24 hours, that observes equal number has nuclear (part differentiation) cell and blank (a breaking up) cell.Getting about 0.5ml equal portions in this stage stores.Cultivate after 48 hours, observe the rectangle cell of a few tentacle (strand) with the DMEM culture medium.Yet, when adopting the RPMI culture medium, observe many difform nucleated cell.Cultivate and find after 48 hours that vessel volume is filled to 37ml graticule place.Getting the 0.5ml equal portions in this stage stores.After 72 hours, the long tentacle of observing cell is crosslinked, similar nervous tissue with the DMEM cultivation.The RPMI culture medium cultivate produced several minicells and several around central cell cumulative cell.Getting the 0.5ml equal portions in this stage stores.

The embryonic cell equal portions that to cultivate in containing the culture medium of culture add in the more many cells culture medium (RPMI or DMEM) that contains other fractions tested that is contained in the sterile chamber (Tarsons steriflask).Under room temperature, carbon dioxide environment, cultivate this stem cell different time, promptly 18 hours-5 days with horizontal level.Cultivate after 24 hours, once more about 0.5ml equal portions are added in more culture medium, cultivate again.Get equal portions and prepare the instant compositions, and be used to store the cell of different breeding phases.Some experiment conditions of being followed when table 46 is described amplification hES cell.

Embodiment 2

Complete or broken embryo is suspended in the culture medium.In addition, add 84 μ l progesterone and 84 μ l β hCG.Test contains the pollution condition of the culture medium of embryonic cell, discards infected embryo.Embryonic cell amplification and storage with this form.

Cultivate after 1 day, the culture medium adding that 1ml is contained embryonic cell is contained in the 46ml cell culture medium (DMEM or RPMI) that contains progesterone and β hCG in the 50ml container.Under the environment of room temperature, carbon dioxide, cultivate this stem cell with the upright position.Table 47 shows the example of a few thing variable that this experiment is used.

Cultivate after 24 hours, the ratio of observing nucleated cell (part differentiation) and blank cell (not differentiation) is about 1:4.Cultivate after 48 hours, the ratio of observing nucleated cell and blank cell is 1:2.Getting the 0.5ml equal portions 24 hours and 48 hour stage stores.

The 0.5ml embryonic cell that to cultivate in containing the culture medium of culture adds in the 46ml cell culture medium that contains other fractions tested (RPMI or DMEM) that is contained in the sterile chamber (Tarsons steriflask).Under room temperature, carbonated environment, cultivate this stem cell with horizontal level.Cultivate after 24 hours, once more about 0.5ml equal portions are added in the 46ml culture medium, cultivate again.Some experiment conditions of being followed when table 48 is described amplification hES cell.

Behind 24 hours breeding phases, get equal portions and prepare instant compositions and storage.

Embodiment 3: store

In order to store, the hES cell is added in the 0.5ml memotron, add frozen dose of 16 μ l, as 0.2%DMSO, be stored in-20 ℃ after the gentle concussion.Allow this cell thaw naturally, to prepare the instant compositions or to be used for further amplification.The thaw vigor of cell of detection finds that the 64%-84% cell has vigor.Regularly detect pollution condition and the vigor that stores cell.Table 49 is presented at the vigor that stores the actual parameter of being followed in five kinds of different experiments and thaw the back cell.

Table 49

Numbering	Suspension cell content	DMSO content	DMSO ％	Used culture medium	The vigor of cell thaws
						1	0.5ml	16μl	0.2	NaCl	74％
2	0.5ml	32μl	1.4	NaCl	66％
						3	0.5ml	64μl	0.4	NaCl	70％
4	0.5ml	48μl	0.2	NaCI	78％
						5	0.5ml	16μl	0.2	NaCl	88％

Embodiment 4

Carry out pollution or the infection or unusual that embryonic cell is checked in various detections in amplification and storage stage with different interval, and identify the hES derivant that exists in each culture.Described different the detection comprises HIV, the detection of HbSAg (hepatitis), be used for the conspicuous conventional PCR (tuberculosis) that detects (Kochs test) of section, divide band CG method to carry out chromosome analysis with the wooden Sa of Ji, detect bilirubin by Ji-Ge Fa (Jendrasiik and Grofmethod), detect albumin (hepatic progenitor cells) with the BCG dye binding method, detect insulin (pancreatic progenitor cell) by the CLIA method, detect neurofilament (neuron progenitor cell) by SABC, detect CD34 (hemopoietic progenitor cell) by SABC, by PNPP method detection of alkaline phosphatase (undifferentiated cell), histopathology (by the identification of morphology cell) etc.Detect condition of culture by manual culture plate method and manual sensitivity, identify fungal infection with versatrek/AP1.Carry out all these detections, check cell counting and cell viability in each stage.Table 50 provides the result of some detections

Embodiment 5: the hES cell that preparation is used to transplant

Get the 15ml stem cell suspension, centrifugal 7 minutes of 1000r.p.m..Abandoning supernatant.2-15ml saline is added in the precipitation, thereby suspend this stem cell.Check the microbial contamination of this suspension.Also carry out the vigor inspection.

Embodiment 6: promptly to store the hES cell with form

Labelling is full of the container (syringe, test tube, culture bottle, bottle) of transplantation hES cell suspension, is stored in-20 ℃.Keep cold chain to the transplanting stage.Before facing transplanting, allow this suspension thaw naturally.

Cell line

Repeat subculture more than 100 times, to set up hES cell of the present invention.In addition, detect the pollution/infection conditions of subculture thing in each stage of amplification or storage.Discovery hES cell line in the subculture cycle more than 5 years is stable, and does not have any unusual.

The scope of the invention is not subjected to the restriction of the specific embodiment and embodiment, and they are intended to illustrate many aspects of the present invention, and any embodiment that is equal on the function all falls into the scope of the invention.It will be understood by those skilled in the art that maybe and can determine that experiment that need not be too much just can obtain many equivalents of the specific embodiment of the invention as herein described.Other equivalents of these and all should fall into the scope of appended claims.All patents, the patent application that this paper is quoted and deliver thing to include this paper in full in for referencial use.

Claims (117)

1. pharmaceutical composition for the treatment of disease, imbalance or disease, it comprises the people embryo who treats effective dose and does (hES) cell and/or its derivant, wherein said hES cell and/or its derivant do not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, fibroblast growth factor, film be in conjunction with steel factor or solubility steel factor or conditioned medium, and wherein said hES cell and/or its derivant are suspended in the pharmaceutically acceptable biocompatible solution.

2. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions is for promptly using form.

3. pharmaceutical composition as claimed in claim 2 is characterized in that, described is pre-filled syringe with form promptly.

4. pharmaceutical composition as claimed in claim 2 is characterized in that, described stem cell has treatment and goes up effectively enough vigor.

5. pharmaceutical composition as claimed in claim 4 is characterized in that the vigor of described stem cell is greater than 40%.

6. pharmaceutical composition as claimed in claim 1 is characterized in that described biocompatible solution is a saline.

7. pharmaceutical composition as claimed in claim 1 is characterized in that, described biocompatible solution also comprises antimicrobial, antibacterial or other medicament.

8. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the hematopoietic stem cell CFU-GM for the treatment of effective dose.

9. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the neuronal stem cell CFU-GM for the treatment of effective dose.

10. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the hematopoietic stem cell CFU-GM and the neuronal stem cell CFU-GM for the treatment of effective dose.

11. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the mescenchymal stem cell CFU-GM for the treatment of effective dose.

12. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the insulin-producing stem cell CFU-GM for the treatment of effective dose.

13. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the hepatocyte stem cell CFU-GM for the treatment of effective dose.

14. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the cardiac stem cells CFU-GM for the treatment of effective dose.

15. pharmaceutical composition as claimed in claim 1 is characterized in that, described compositions comprises the epithelial stem cell CFU-GM for the treatment of effective dose.

16. pharmaceutical composition as claimed in claim 8 is characterized in that, the treatment effective dose of hematopoietic stem cell CFU-GM is about 750 in the described compositions, 000-160,000,000 cell.

17. pharmaceutical composition as claimed in claim 9 is characterized in that, the treatment effective dose of neuronal stem cell CFU-GM is about 750 in the described compositions, 000-160,000,000 cell.

18. pharmaceutical composition as claimed in claim 1 is characterized in that, the treatment effective dose of described hES cell and/or its derivant about 750 in about 0.25-100ml biocompatible solution, 000-160,000,000 cell.

19. pharmaceutical composition as claimed in claim 1 is characterized in that, the treatment effective dose of described hES cell and/or its derivant about 750 in about 0.25-100ml biocompatible solution, 000-80,000,000 cell.

20. pharmaceutical composition as claimed in claim 1, it is characterized in that described disease, imbalance or disease are selected from: cancer, apoplexy, genetic diseases, hepatopathy, disorder of development, degenerative disease, familial disease or neural traumatic disease, angiopathy, dermatosis or imbalance, autoimmune disease, oculopathy, nephropathy, heart disease, flesh bone disease, reproduction or fertility disease, arthritis or hematopathy.

21. pharmaceutical composition as claimed in claim 1, it is characterized in that described disease, imbalance or disease are selected from: acute myeloid leukemia, adenocarcinoma, arthritis, astrocytoma, the auditory nerve atrophy, autism, autoimmune disease, Alzheimer's disease, ankylosing spondylitis, becker's dystrophy, brain injury, burn, cerebrovascular trauma, the cerebral palsy, stupor, corneal ulcer, corneal graft rejection, neural cortex-substrate degeneration, coronary heart disease, diabetes, dull-witted, mongolism, Duchenne's dystrophy, end-stage renal disease, erb's palsy, face shoulder muscular dystrophy, fertility is sick, Friedreich ataxia, heart failure, hepatocarcinoma, heritability dynamoneure disease, Huntington chorea, krabbe's disease, limb-girdle muscular dystrophy, liver cirrhosis, degeneration of macula, mental retardation, multiple sclerosis, motor neuron, myocardial infarction, nephrotic syndrome, Niemann-Pick disease, the disunion skin ulcer, olivopontocerebellar atrophy, optic atrophy, parkinson disease, electric shock back encephalopathy, encephalopathy behind the rabies vaccine, decubital ulcer, progressive supranuclear plasy, psoriasis, phthisis bulbi, restrictive cardiomyopathy, retinitis pigmentosa, right bundle branch block, sarcoidosis, sinus bradycardia, tumor of spinal cord, spinal cord muscular dystrophy, spinocerebellar ataxia, Stevens Johnson syndrome, systemic lupus erythematosus (sle), thrombocytopenia, thalassemia, ulcerative colitis, vegetative state, cystic fibrosis, interstitial lung disease, azoospermia, primary ovarian failure, aphthous ulcer, hormonal imbalance, osteoarthritis, Horner syndrome and osteogenesis imperfecta, passage disease or hypogammaglobulinemia.

22. compositions that comprises hES cell and/or its derivant, described hES cell and/or its derivant do not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, fibroblast growth factor, film be in conjunction with steel factor and/or solubility steel factor or conditioned medium, and wherein said hES cell and/or its derivant are wrapping in biocompatibility, differential permeability structure or the substrate.

23. compositions as claimed in claim 22 is characterized in that, described hES cell and/or its derivant comprise the hematopoietic stem cell CFU-GM.

24. compositions as claimed in claim 22 is characterized in that, described hES cell and/or its derivant comprise the neuronal stem cell CFU-GM.

25. compositions as claimed in claim 22 is characterized in that, described hES cell and/or its derivant comprise the combination of hematopoietic stem cell and neuronal stem cell CFU-GM.

26. compositions as claimed in claim 22, it is characterized in that described biocompatibility, differential permeability structure or substrate are selected from biopolymer, polypeptide, protein, polysaccharide, fibronectin, collagen, laminin, keratin, fibrin, Fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose or gelatin.

27. compositions as claimed in claim 22 is characterized in that, described hES cell and/or its derivant are wrapping in the mixture of the mixture of agarose and collagen or agarose and gelatin.

28. a method of separating the hES cell, described method comprises:

(a) with 2-7 age in days embryo collection in minimum essential medium,

(b) separate described embryo's hES cell by mechanical means.

29. method as claimed in claim 28 is characterized in that, described embryo is instar embryo on the 2nd.

30. method as claimed in claim 28 is characterized in that, described mechanical means are concussions.

31. the method for the hES cell that increases, described hES cell does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, said method comprising the steps of:

(a) the hES cell is added in the cell culture medium of being made up of minimum essential medium, progestogen and β-human chorionic gonadotropin (β hCG) agonist; With

(b) in the environment of about 34-38 ℃ temperature, about 3.5-6% carbon dioxide, cultivated described stem cell about 12-48 hour.

32. the method for the described hES cell of the claim 31 that increases is characterized in that described cell culture medium is RPMI.

33. the method for amplification hES cell as claimed in claim 31, described method comprises:

(c) get the stem cell equal portions that step (b) is cultivated, wherein said equal portions contain at least a stem cell,

(d) described cell is resuspended in the cell culture medium that contains progesterone and β hCG, this cell of five equilibrium,

(e) with the cell culture medium that does not contain progestogen and β hCG agonist dilute described cell and

(f) in the environment of about 34-38 ℃ temperature, about 3.5-6% carbon dioxide, cultivated described cell about 12-48 hour.

34. the method for the described hES cell of the claim 31 that increases is characterized in that described cultivation is carried out in the water leg cell culture incubator.

35. the method for the described hES cell of the claim 31 that increases is characterized in that, under obvious anoxybiotic condition, cultivates described stem cell with culture medium in the biocompatibility container.

36. the method for the described hES cell of the claim 35 that increases is characterized in that, described stem cell does not break up when propagation.

37. the method for the described hES cell of the claim 31 that increases is characterized in that, used hES cell is isolating by instar embryo on the 3rd by mechanical means.

38. the method for the described hES cell of the claim 31 that increases also comprises the step of the pollution condition of the stem cell that detects amplification.

39. the method for the described hES cell of the claim 31 that increases is characterized in that described incubation is carried out in the biocompatibility container.

40. the method for the described hES cell of the claim 31 that increases is characterized in that, the stem cell equal portions of described cultivation and the ratio of cell culture medium are about 1:3.5-1:35.

41. the method for the described hES cell of the claim 31 that increases is characterized in that, described cultivation is obviously being carried out in the anoxic environment.

42. the method for the described hES cell of the claim 31 that increases is characterized in that, described cultivation is almost completely occupied by culture medium and keeps carrying out in the vertical biocompatibility container at volume.

43. method that the hES cell is partly broken up, described hES cell does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with steel factor, solubility steel factor and conditioned medium, said method comprising the steps of:

(a) the hES cell is added in the cell culture medium of being made up of minimum essential medium; With

(b) in the environment of about 34-38 ℃ temperature, about 3.5-6% carbon dioxide, cultivated described stem cell about 12-48 hour.

44. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that, described cell culture medium is RPMI or DMEM.

45. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that described cultivation is carried out in the water leg cell culture incubator.

46. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that, in the biocompatibility container, cultivates the described stem cell in the culture medium under the condition of obvious aerobic.

47. the method that the hES of making cell as claimed in claim 46 partly breaks up is characterized in that, the differentiation when propagation of described stem cell.

48. the method that the hES of making cell as claimed in claim 43 partly breaks up also comprises the step of the pollution condition of the stem cell that detects amplification.

49. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that described incubation is carried out in the biocompatibility container.

50. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that, the stem cell equal portions of described cultivation and the ratio of described cell culture medium are about 1:3.5-1:35.

51. the method that the hES of making cell as claimed in claim 43 partly breaks up is characterized in that, described cultivation is to carry out in the environment of obvious aerobic.

52. the method that the hES of making cell as claimed in claim 51 partly breaks up is characterized in that described cultivation is carried out in the biocompatibility container, described container keeps horizontal level.

53. a method for preparing the instant preparation that is used for hES cell that the people transplants and/or its derivant, described method comprises:

(a) obtain not contain animal product, raise cell, somatomedin, leukaemia inhibitory factor, the combination of complementarity mineral, amino acid supplements, vitamin replenisher, fibroblast growth factor, film be in conjunction with the hES cell of steel factor, solubility steel factor and conditioned medium

(b) centrifugal described stem cell obtain precipitation and

(c) described precipitation is suspended in the biocompatible solution.

54. preparation as claimed in claim 53 is used for the method for the instant preparation of hES cell that the people transplants and/or its derivant, also comprises:

(d) described preparation is stored in approximately-15 to-72 ℃ and

(e) allow the preparation of described storage thaw naturally before facing transplanting,

The vigor of described cell is at least 40% after thawing.

55. be used for the method for the instant preparation of hES cell that the people transplants and/or its derivant as claim 53 or 54 described preparations, also be included in the pollution condition that detects described preparation before transplanting.

56. preparation as claimed in claim 53 is used for the method for the instant preparation of hES cell that the people transplants and/or its derivant, it is characterized in that, the described hES cell and/or its derivant that obtain in (a) produce by claim 31 or 45 described methods.

57. one kind is stored in method under the condition of can living with hES cell and/or its derivative formulations, described method comprises:

(a) the weighting profit requires the hES cell of 31 or 45 described method preparations and/or the preparation of its derivant,

(b) add frozen dose and

(c) with described cell cryopreservation in-15 to-72 ℃ approximately.

58. a method that stores the described hES cell of claim 57 and/or its derivative formulations is characterized in that, the described frozen dose of ratio with culture medium content is about 1:500-16:1000.

59. a method that stores the described hES cell of claim 57 and/or its derivative formulations is characterized in that described storage is to be stored in the biocompatibility container.

60. a method that stores the described hES cell of claim 57 and/or its derivative formulations is characterized in that, described cell cryopreservation is in-18 to-20 ℃ approximately.

61. the method for the disease of a treatment target, imbalance or disease, described method comprises hES cell and/or its derivant that gives described object treatment effective dose, and described cell does not contain animal product, raises cell, somatomedin, leukaemia inhibitory factor, fibroblast growth factor or conditioned medium.

62. method as claimed in claim 61 is characterized in that, described hES cell and/or its derivant produce with claim 31 or 45 described methods.

63. method as claimed in claim 61, it is characterized in that described disease, imbalance or disease are selected from: cancer, apoplexy, genetic diseases, hepatopathy, disorder of development, degenerative disease, familial disease or neural traumatic disease, angiopathy, dermatosis and imbalance, autoimmune disease, oculopathy, nephropathy, heart disease, flesh bone disease, reproduction and fertility disease, arthritis and hematopathy.

64., it is characterized in that described nervous system disease is the developmental character nervous system disease as the described method of claim 63, be selected from autism, cerebral palsy, erb's palsy, mental retardation or progressive supranuclear plasy.

65. as the described method of claim 63, it is characterized in that, described nervous system disease is the nervous system degeneration disease, is selected from Alzheimer's disease, cortex-substrate degeneration, deafness (auditory nerve atrophy), dementia, Friedreich ataxia, motor neuron, multiple sclerosis, olivopontocerebellar atrophy, parkinson disease or spinocerebellar ataxia.

66., it is characterized in that described nervous system disease is a nervous system injury as the described method of claim 63, be selected from encephalopathy behind encephalopathy after brain injury, stupor, the electric shock, the rabies vaccine, spinal cord injury or injury and vegetative state.

67., it is characterized in that described nervous system disease is cerebrovascular trauma or apoplexy as the described method of claim 63.

68., it is characterized in that described nervous system disease is a familial disease as the described method of claim 63, be selected from heritability dynamoneure disease or Huntington chorea.

69., it is characterized in that described hepatopathy and nephropathy are selected from as the described method of claim 63: liver cirrhosis, latter stage nephropathy, nephrotic syndrome or Niemann-Pick disease.

70., it is characterized in that described dermatosis is selected from as the described method of claim 63: arthritis, arteriosclerosis, burn, disunion ulcer, decubital ulcer, psoriasis, systemic lupus erythematosus (sle) or sarcoidosis.

71., it is characterized in that described autoimmune disease is selected from as the described method of claim 63: thrombocytopenia, systemic lupus erythematosus (sle), sarcoidosis or ulcerative colitis.

72., it is characterized in that described genetic diseases is selected from as the described method of claim 63: mongolism, ankylosing spondylitis, thalassemia or Huntington chorea.

73., it is characterized in that described oculopathy is selected from as the described method of claim 63: optic atrophy, phthisis bulbi, degeneration of macula, retinitis pigmentosa, corneal abrasion, corneal graft rejection or corneal ulcer.

74., it is characterized in that described flesh bone disease is selected from as the described method of claim 63: Duchenne's dystrophy, face shoulder muscular dystrophy, limb-girdle muscular dystrophy, spinal cord muscular dystrophy or becker's dystrophy.

75., it is characterized in that described heart disease is selected from as the described method of claim 63: myocardial infarction, right bundle branch block, restrictive cardiomyopathy, heart failure, sinus bradycardia or coronary heart disease.

76., it is characterized in that described cancer is selected from as the described method of claim 63: acute myeloid leukemia, adrenal gland's adenocarcinoma, astrocytoma, hepatocarcinoma or tumor of spinal cord.

77., it is characterized in that described disease is diabetes as the described method of claim 63.

78. a treatment suffers from disease, the method of the object of imbalance or disease, described method comprises by intramuscular injection, intravenous injection, epidural injection, epidural catheter, retrobulbar injection, subcutaneous injection, intracardiac injection, intracapsular injection, intrathecal injection, local application, use in the damage or intravenous infusion, or pass through aerosol apparatus, spraying, the intravaginal approach, partial eye drop and auristillae are treated hES cell and/or its derivant of cultivating in the culture medium of effective dose, and described culture medium does not contain animal product, raise cell, conditioned medium, somatomedin, leukaemia inhibitory factor, fibroblast growth factor, film is in conjunction with steel factor or solubility steel factor.

79., it is characterized in that described hES cell and/or its derivant produce with claim 31 or 45 described methods as the described method of claim 78.

80. described treatment suffers from the method for the object of disease, imbalance or disease as claim 78, it is characterized in that, the treatment effective dose of described hES cell and/or its derivant is about 750,000-160,000,000 cell.

81. described treatment suffers from the method for the object of disease, imbalance or disease as claim 78, it is characterized in that, described hES cell and/or its derivant comprise hematopoietic stem cell CFU-GM, neuronal stem cell CFU-GM, mescenchymal stem cell CFU-GM, epithelial stem cell CFU-GM, kidney stem cell CFU-GM, cardiac stem cells CFU-GM or liver stem cells CFU-GM, or its combination.

82. described treatment suffers from the method for the object of disease, imbalance or disease as claim 78, it is characterized in that described disease is selected from cancer, apoplexy, genetic diseases, hepatopathy, disorder of development, degenerative disease, familial disease or neural traumatic disease, angiopathy, dermatosis and imbalance, autoimmune disease, oculopathy, nephropathy, heart disease, flesh bone disease, reproduction and fertility disease, arthritis or hematopathy.

83. described treatment suffers from disease as claim 78, the method of the object of imbalance or disease, it is characterized in that described disease, imbalance or disease are selected from: acute myeloid leukemia, adenocarcinoma, arthritis, astrocytoma, the auditory nerve atrophy, autism, autoimmune disease, Alzheimer's disease, ankylosing spondylitis, becker's dystrophy, brain injury, burn, cerebrovascular trauma, the cerebral palsy, stupor, corneal ulcer, corneal graft rejection, neural cortex-substrate degeneration, coronary heart disease, diabetes, dull-witted, mongolism, Duchenne's dystrophy, end-stage renal disease, erb's palsy, face shoulder muscular dystrophy, fertility is sick, Friedreich ataxia, heart failure, hepatocarcinoma, heritability dynamoneure disease, Huntington chorea, krabbe's disease, limb-girdle muscular dystrophy, liver cirrhosis, degeneration of macula, mental retardation, multiple sclerosis, motor neuron, myocardial infarction, nephrotic syndrome, Niemann-Pick disease, the disunion skin ulcer, olivopontocerebellar atrophy, optic atrophy, parkinson disease, electric shock back encephalopathy, encephalopathy behind the rabies vaccine, decubital ulcer, progressive supranuclear plasy, psoriasis, phthisis bulbi, restrictive cardiomyopathy, retinitis pigmentosa, right bundle branch block, sarcoidosis, sinus bradycardia, tumor of spinal cord, spinal cord muscular dystrophy, spinocerebellar ataxia, Stevens Johnson syndrome, systemic lupus erythematosus (sle), thrombocytopenia, thalassemia, ulcerative colitis, vegetative state, cystic fibrosis, interstitial lung disease, azoospermia, primary ovarian failure, aphthous ulcer, hormonal imbalance, osteoarthritis, Horner syndrome and osteogenesis imperfecta, passage disease or hypogammaglobulinemia.

84. described treatment suffers from the method for the object of disease, imbalance or disease as claim 78, it is characterized in that, the described hES of giving cell and/or its derivant can not cause tumor, teratoma or chromosomal change.

85. the method for the spinal cord injury of a treatment target (SCI), described method comprises:

A) give by subcutaneous injection about 750,000-80,000,000 hES cell and/or its derivant;

B) back repeating step (a) is at the fixed time treated hES cell and/or its derivant of effective dose then by intramuscular injection;

C) treat hES cell and/or its derivant of effective dose by intravenous injection or infusion, wherein said cell comprises neuronal stem cell CFU-GM and hematopoietic stem cell CFU-GM;

D) treat hES cell and/or its derivant of effective dose by epidural injection, wherein said cell comprises the neuronal stem cell CFU-GM, and described dosage is repeated to give according to status of patient clinical and/or the neurologic examination evaluation in the back at the fixed time;

E) treat hES cell and/or its derivant of effective dose by the tail side injection, wherein said cell comprises the neuronal stem cell CFU-GM;

F) treat hES cell and/or its derivant of effective dose by intrathecal injection or subarachnoid block conduit, wherein said cell comprises the neuronal stem cell CFU-GM;

G) treat hES cell and/or its derivant of effective dose by epidural injection or epidural catheter, wherein said cell comprises the neuronal stem cell CFU-GM;

H) treat hES cell and/or its derivant of effective dose at the spinal column either side by degree of depth spinal cord injection; With

I) treat hES cell and/or its derivant of effective dose by intravenous infusion;

Wherein carry out step (a) and (b) earlier, all the other steps can be carried out in random order.

86., also comprise repeating step (f) as the described method of claim 85, be step (g) then, reveal the clinical sign of recovering by described SCI up to this Object table.

87., it is characterized in that described hES cell and/or its derivant produce with claim 31 or 45 described methods as the described method of claim 85.

88. as the described SCI Therapeutic Method of claim 85, it is characterized in that, in step (a) and the step (b) about 750,000-80,000,000 hES cell and/or its derivant are suspended in the 0.25-1.0ml biocompatible solution, and wherein said cell comprises hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM.

89. as the described SCI Therapeutic Method of claim 85, it is characterized in that, in the step (f) about 750,000-11,000,000 hES cell and/or its derivant are suspended in the 2.0-4.0ml biocompatible solution, and wherein said cell comprises hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM.

90. as the described SCI Therapeutic Method of claim 85, it is characterized in that, in the step (g) about 750,000-80,000,000 hES cell and/or its derivant are suspended in the 15-40ml biocompatible solution, and wherein said cell comprises hematopoietic stem cell CFU-GM and neuronal stem cell CFU-GM.

91., it is characterized in that described SCI patient's treatment causes decubital ulcer to be improved as the described method of claim 85.

92. method for the treatment of developmental character, degeneration, familial and traumatic nervous system disease and cerebrovascular disease, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intrathecal injection, epidural catheter infusion or subarachnoid block conduit infusion and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

93. the dermopathic method of treatment, described method comprise give by subcutaneous or intravenous injection about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

94., it is characterized in that described hES cell and/or CFU-GM are mixed as the described Therapeutic Method of claim 93 in preparing to be applied to the biological compatibility carrier of injured skin.

98., it is characterized in that described biological compatibility carrier is gel, ointment, paste or aerosol spray as the described Therapeutic Method of claim 94.

95. a method for the treatment of decubital ulcer, described method comprise by part or local application and give by intramuscular injection about 750,000-80,000,000 hES cell and/or its derivant.

96. a method for the treatment of autoimmune disease, described method comprise give by intramuscular injection, intravenous injection, subcutaneous injection, intra-articular injection or intravenous infusion or its combination about 750,000-160,000,000 hES cell and/or its derivant.

97. method for the treatment of genetic diseases, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intrathecal injection, epidural catheter infusion or subarachnoid block conduit infusion or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

98. a method for the treatment of gangrene, described method comprise by intravenous injection, intramuscular injection, living tissue and extremely organize the junction local application or its combination give about 750,000-160,000,000 hES cell and/or its derivant.

99. the method for the aging-related disease of treatment, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, local application about 750,000-160, suspension or mixture that 000,000 hES cell and/or its derivant form in biological compatibility carrier.

100., it is characterized in that described biological compatibility carrier is gel, ointment, paste or aerosol spray as the described Therapeutic Method of claim 102.

A kind of method for the treatment of diabetes, described method comprise give by intravenous or intramuscular injection or the two about 750,000-160,000,000 people embryo insulin-producing CFU-GM.

A kind of method for the treatment of the cardiovascular diseases, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intracardiac injection, angiography or direct injection or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

Method as the described treatment of claim 103 cardiovascular diseases is characterized in that, describedly carries out during angiography.

A kind of method for the treatment of hepatopathy and nephropathy, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intravenous infusion or local injection or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise the hematopoietic stem cell CFU-GM, produce albumin stem cell CFU-GM and produce bilirubin stem cell CFU-GM.

A kind of method for the treatment of fertility and reproductive disease, described method comprise by injection in local intramuscular injection, the testis, near the epididymis subcutaneous injection or its combination give about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the hematopoietic stem cell CFU-GM.

A kind of method for the treatment of the flesh bone disease, described method comprises by intravenous injection, subcutaneous injection, intramuscular injection, intravenous catheter infusion or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

A kind of method for the treatment of oculopathy, described method comprises by local intravenous injection, subcutaneous injection intramuscular injection, retrobulbar injection, intravitreal injection or local application or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM, hematopoietic stem cell CFU-GM and/or mescenchymal stem cell CFU-GM.

Method as the described treatment oculopathy of claim 107 is characterized in that, give by retrobulbar injection about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.

Method as the described treatment oculopathy of claim 107 is characterized in that, give by intravitreal injection about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprises the neuronal stem cell CFU-GM.

110. the method as the described treatment oculopathy of claim 107 is characterized in that, with about 750, and 000-160,000,000 hES cell and/or its derivant are applied to contact lens with the treatment corneal abrasion, and wherein said cell comprises the mescenchymal stem cell CFU-GM.

111. method for the treatment of pneumonopathy, described method comprises by intramuscular injection, intravenous injection, spraying, aerosol apparatus or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

112. method for the treatment of the hormone disease, described method comprises by intramuscular injection or intravenous injection or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

113. method for the treatment of aphthous ulcer and other ulcer, described method comprises by intramuscular injection or intravenous injection or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

114. method for the treatment of the osteoarthritis of knee joint and hip joint, described method comprises by intramuscular injection, intravenous injection, intra-articular injection or its combination and gives about 750,000-160,000,000 hES cell and/or its derivant, wherein said cell comprise neuronal stem cell CFU-GM and/or hematopoietic stem cell CFU-GM.

115. a detection compound is to the method for the effect of hES cell and/or its derivant, described method is included in this chemical compound existence and cultivates hES cell and/or its derivant that obtains with claim 31 or 45 described methods down and measure the effect of this chemical compound to described cell.

116. method that medicine is delivered to object, described method is included in this medicine and has hES cell and/or its derivant of cultivating down with claim 31 or 45 described methods acquisitions, wherein said cell is taken in this medicine, give the site of described object with described cell, this medicine is delivered to this site.